The authors would like to thank the Department of Biotechnology, Government of India, for financial support.

1. Growth of microbial strains
<ol>
	<li>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.</li>
	<li>Similarly, prepare two more flasks of 100 mL of Luria Broth and place cotton plugs on the neck of the flasks.</li>
	<li>Cover the cotton plugs with aluminum foil and autoclave the flasks for 15 min at 121 &#176;C and 15 psi to sterilize the media.</li>
	<li>After autoclaving, let the media cool down to room temperature.</li>
	<li>For the preparation of primary culture of a strain, pick a single colony from an LA plate using an inoculation loop and inoculate in a test tube containing 5 mL of sterile Luria broth.</li>
	<li>Incubate the test tube overnight at 30 &#176;C at 180 rpm.</li>
	<li>Inoculate the flasks containing 100 mL of autoclaved Luria Broth by adding 1 mL of overnight grown seed cultures to the flasks inside the laminar air flow cabinet.</li>
	<li>Incubate the flasks in a rotary incubator at 30 &#176;C and 180 rpm for 7 days.</li>
	<li>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 x g for 20 min in a refrigerated centrifuge at 4 &#176;C.</li>
	<li>Gently, pour the cell free supernatant into a fresh beaker and use it in screening assays for biosurfactant production.</li>
</ol>
2. Screening assays for biosurfactant production
NOTE: In the following sections, commercial surfactant (Saponin) was used as a positive control while water and uninoculated media were used as a negative control.
<ol>
	<li>Drop collapse assay
	<ol>
		<li>Take a clean glass slide and coat the surface of the slide with 200 &#181;L of oil.</li>
		<li>Add 20 &#181;L of the cell free supernatant to the center of the oil and leave it undisturbed for 2-3 min. 
		NOTE: If the drop collapses, score the supernatant positive for the presence of biosurfactant.</li>
	</ol>
	</li>
	<li>Oil spreading assay
	<ol>
		<li>Take 20 mL of double distilled water in a Petri plate (75 mm diameter) and add 200 &#181;L of crude oil to the surface of the water.</li>
		<li>Add 20 &#181;L of cell free supernatant to the center of the oil and leave it undisturbed for 1 min. 
		NOTE: If a clearing zone is formed because of oil displacement, score the supernatant positive for the presence of the biosurfactant.</li>
	</ol>
	</li>
	<li>Emulsion index assay (E24 assay)
	<ol>
		<li>Add 4 mL of petrol (gasoline) and cell-free supernatant each into a clean glass test tube.</li>
		<li>Vortex the mixture vigorously for 3 min and leave it undisturbed for the next 24 h.</li>
		<li>After 24 h, determine the E24 index as a percentage of the height of the emulsified layer (cm) with respect to the height of the entire liquid column (cm). 
		<img alt="Equation 1" src="/files/ftp_upload/63207/63207eq01.jpg" style="margin: auto;" /> 
		NOTE: If an emulsion (oil in water or water in oil) is observed after 24 h, the supernatant is likely to contain the biosurfactant.</li>
	</ol>
	</li>
	<li>Surface tension measurement 
	NOTE: The surface tension was measured using the Du No&#252;y ring method17. The instrument used in this experiment (see Table of Materials) is very sensitive, so ensure proper cleaning of the glass vessel and the probe.
	<ol>
		<li>Turn on the system and double click on the associated software to open it.</li>
		<li>Clean the glass vessel with the liquid whose surface tension is to be determined.</li>
		<li>Add the liquid (40 mL) into the vessel and mount the vessel on the vessel holder.</li>
		<li>Unlock the probe holder and mount the probe on it. Now lock the probe holder by pressing the Lock button on the manual controller.</li>
		<li>Using the manual controller, adjust the height of the platform such that the probe is around 2-3 mm away from the surface of the liquid.</li>
		<li>Now use the software to measure the surface tension. Click on File located on the top left panel of the screen. Click on Open Workspace. A pop-up window will appear.</li>
		<li>Scroll down and double-click on the K100: Surface and Interfacial Tension icon.</li>
		<li>Now, click on the File icon located in the top-left corner of the screen. Click on New Database. Enter the name for saving the data and click on OK.</li>
		<li>Again, click on File &#62; New Measurement &#62; SFT &#62; Ring. Enter the name for the measurement. Ensure that the configuration template shows SFT ring.</li>
		<li>Fill in the details in the Measurement Configuration window by selecting the probe and the vessel that is being used for the measurement. Also, fill in the details of the liquid and the gas phase. 
		NOTE: The liquid phase will be water, while the gas phase will be air. The density of the liquid phase is the density of the cell free supernatant. This can be determined by taking the weight of 50 mL of the liquid and calculating density as Kg/m3.</li>
		<li>Now click on the Procedure tab and fill in the following details: Detection Speed: 6 mm/min, Detection Sensitivity: 0.005 g, Search Speed: 6 mm/min, Search Sensitivity: 0.005 g, Measuring Speed: 3 mm/min, Measuring Sensitivity: 0.001 g, Immersion Depth: 3 mm, Return Distance: 10%, correction: Harkins &#38; Jordan, max values: 5. Click on OK.</li>
		<li>In the pop-up window, select the database to store data and click on OK. 
		NOTE: One can create a new database here or add new measurements to the existing data.</li>
		<li>Now click on the Play button located on the top-center of the screen. The system will start executing the script. After the system stabilizes, it will detect the surface. Immerse the probe into the liquid, move the probe back and forth and detect the tension in the formed lamella.</li>
		<li>For obtaining the results, click on the Measurement icon on the middle-left side of the screen. Click on Data and note down the surface tension determined.</li>
		<li>After completion of the measurement, lower the height of the platform and unlock and unmount the probe and the vessel from the instrument. 
		NOTE: To determine the decrease in the surface tension due to biosurfactant production, uninoculated LB should be used as a control.</li>
	</ol>
	</li>
</ol>
3. Biosurfactant extraction
<ol>
	<li>Adjust the pH of the cell free supernatant to 2 using 2 N HCl. Store the mixture at 4 &#176;C overnight.</li>
	<li>Add equal volume of chloroform-methanol mixture (2:1) to the supernatant and mix vigorously for 20 min.</li>
	<li>Leave the mixture undisturbed for phase separation to occur.</li>
	<li>Remove the upper phase containing water and methanol and leave the lower phase containing the biosurfactant to evaporate in a fume hood.</li>
	<li>After evaporation of the organic phase, redissolve the honey-colored crude biosurfactant in 3 mL of chloroform and use this mixture for further identification and characterization of the biosurfactant.</li>
</ol>
4. Emulsion stability studies
<ol>
	<li>Emulsion stability at different temperatures
	<ol>
		<li>Take 5 mL of cell free supernatants in different test tubes.</li>
		<li>Add 5 mL of petrol to each test tube and mix vigorously by vortexing for 3 min.</li>
		<li>Incubate the test tubes overnight in different water baths at different temperatures (30 &#176;C, 40 &#176;C, 50 &#176;C, 60 &#176;C, and 70 &#176;C).</li>
		<li>After 24 h, estimate the emulsion indices as mentioned earlier.</li>
	</ol>
	</li>
	<li>Emulsion stability at different pH values
	<ol>
		<li>Take 5 mL of the cell free supernatant in clean test tubes.</li>
		<li>Adjust the pH of cell free supernatants (2, 4, 6, 8, and 10) using 1 N HCl and 1 N NaOH.</li>
		<li>Add an equal amount of petrol to the test tubes and mix vigorously by vortexing for 3 min.</li>
		<li>Leave the test tubes undisturbed at room temperature for 24 h.</li>
		<li>Estimate the emulsion index as mentioned earlier.</li>
	</ol>
	</li>
	<li>Emulsion stability at different salt concentrations
	<ol>
		<li>Take 5 mL of the cell free supernatant in clean test tubes.</li>
		<li>Add different amounts of salt (NaCl) to the supernatants (0 g/L, 5 g/L, 10 g/L, 20 g/L, 60 g/L and 80 g/L).</li>
		<li>Dissolve the salts in the cell free supernatants by vortexing for 3 min.</li>
		<li>Add an equal amount of petrol to the test tubes and mix vigorously by vortexing for 3 min.</li>
		<li>Leave the test tubes undisturbed at room temperature for 24 h.</li>
		<li>Estimate the emulsion index after 24 h.</li>
	</ol>
	</li>
</ol>
5. Determining the nature of the biosurfactant
<ol>
	<li>TLC of the extracted biosurfactant
	<ol>
		<li>Spot 20 &#181;L of the biosurfactants on TLC plates. Spot 2 &#181;L at one time.</li>
		<li>Spot the biosurfactants on three different TLC plates.</li>
		<li>Prepare a 100 mL mixture of the eluent containing chloroform:methanol (2:1) and add the eluent to the TLC chamber. Close the lid of the chamber and allow it to saturate for 20 min.</li>
		<li>After drying the plates, place the TLC plates inside the chamber saturated with a chloroform methanol mixture and run the TLC.</li>
		<li>After the eluent has reached the top of the TLC plate (1 cm away from the top), take the plates out and let it air dry.</li>
	</ol>
	</li>
	<li>Staining for lipid detection
	<ol>
		<li>Take a clean TLC chamber and add some (5-10) granules of iodine into the fresh chamber and saturate the chamber for 5 to 10 mins.</li>
		<li>Place the TLC plate inside the chamber and observe for the development of the yellow spots. If the spots appear, score the biosurfactant positive for the presence of the lipid component.</li>
	</ol>
	</li>
	<li>Staining for peptide or amino acid detection
	<ol>
		<li>Prepare a ninhydrin solution by dissolving 0.4 g of ninhydrin in 20 mL of butanol. Add 0.6 mL of 100% glacial acetic acid to the mixture.</li>
		<li>Spray the TLC plate with ninhydrin solution and let it air dry for 2 min. Heat the plate at 110 &#176;C and observe the development of the color. 
		NOTE: If the blue spots appear, score the biosurfactant positive for the presence of any peptide chain or amino acid.</li>
	</ol>
	</li>
	<li>Staining for carbohydrate detection
	<ol>
		<li>Prepare a solution of p-anisaldehyde by adding 2 mL of p-anisaldehyde to 48 mL of glacial acetic acid containing 1 mL of H2SO4. Add 0.6 mL of acetic acid to the mixture.</li>
		<li>Spray the mixture evenly on a TLC plate and let it air dry for 2 min.</li>
		<li>Incubate the plate at 110 &#176;C and monitor the development of spots. 
		NOTE: If the green or brown spots appear, score the biosurfactant positive for the presence of any carbohydrates.</li>
	</ol>
	</li>
</ol>
6. Chemical identification of the biosurfactant
<ol>
	<li>LCMS of the biosurfactant
	<ol>
		<li>Dissolve 25 mg of the extracted biosurfactant in 1 mL of chloroform.</li>
		<li>Perform LCMS (in a lock spray configuration with a reference scan frequency of 10 s) using a C18 column.</li>
		<li>Use chloroform:methanol (1:1) as a mobile phase and inject 2 &#181;L of the sample into the column at a flow rate of 0.1 mL/min.</li>
		<li>Set the experimental parameters to: polarity: ES positive, capillary voltage: 3 kV, source temperature: 80 &#176;C, desolvation temperature: 300 &#176;C, desolvation gas flow rate: 7,000 L/h, and trap gas flow rate: 0.40 mL/min.</li>
		<li>Scan the ranges from 100 to 1,200 Da during a detection time of 20 mins and survey the ions in positive ES mode.</li>
		<li>Analyze the m/z values using any mass spectrometry quantitively software.</li>
		<li>For analysis, log into the software.</li>
		<li>Click on Batch Search and enter the list of the masses obtained. Survey the results in a positive charge mode and use M + H and M + Na as the adducts. Maintain the accuracy to 10 PPM and tick on Display Structure.</li>
		<li>Click on Search and from the list of compounds, select the one with the lowest PPM level.</li>
	</ol>
	</li>
	<li>1H NMR of the biosurfactant 
	NOTE: 1H NMR of the biosurfactant was performed using a 400 MHz NMR spectrometer (see Table of Materials).
	<ol>
		<li>Dissolve 5 mg of the biosurfactant in 1 mL of deuterated chloroform(CdCl3).</li>
		<li>Transfer the mixture to an NMR tube. Cap the tube properly and insert the tube in the spanner. Adjust the height of the tube using the adjuster tube.</li>
		<li>Place the tube along with the spanner in the NMR machine and follow the steps mentioned below to get an NMR spectrum.</li>
		<li>To select the sample tube type: sx N, where N is the position where the tube was placed (e.g., sx 13, if the tube was placed at the 13th position) in associated software.</li>
		<li>Type edc and press Enter to create a new folder where data can be stored.</li>
		<li>A pop up will appear. Select the solvent by clicking on CdCl 3 in the list and enter the name of the sample.</li>
		<li>To start the protocol, type &#34;getprosol&#34;; to lock the solvent, type &#34;lock cdcl3&#34;.</li>
		<li>Type &#34;topshim&#34; to shim the sample, and finally type &#34;rga;zgefp&#34; for acquiring the data. This will start the protocol.</li>
		<li>After the spectra has been obtained, type &#34;apk;abs n&#34; and press enter for phase and baseline correction.</li>
		<li>To select primary peaks, type &#34;pp&#34; and press Enter. To select only intense peaks, enter &#34;mi&#34; and type in the intensity above which the peaks should be selected. The default value will be 0.2.</li>
		<li>To integrate the peaks, click on Integrate and place the cursor on the left side of the peak to be integrated and while holding the cursor click and drag the cursor around the peak.</li>
		<li>Save the data by clicking on File on the top-left corner, and then click on Save.</li>
		<li>The sample can be ejected from the machine by typing &#34;sx ej&#34;.</li>
		<li>Analyze the peaks and determine the environment of the H atoms.</li>
	</ol>
	</li>
	<li>Fourier Transform Infrared Spectroscopy of the biosurfactant 
	NOTE: FT-IR of extracted biosurfactant was performed using a commercially available spectrophotometer in ATR mode (see Table of Materials).
	<ol>
		<li>Turn the spectrophotometer on and check the purge, desiccant, and detector.</li>
		<li>To collect a spectrum, first collect the background spectrum without a sample in place.</li>
		<li>Take the extracted biosurfactant and dry it completely. Place the dried biosurfactant directly over the diamond crystal, apply pressure and press the ATR touch point.</li>
		<li>In the software, select the number of the scans (enter 30) and scan the spectrum from 400 cm-1 to 4,000 cm-1.</li>
		<li>Click on OK to add the sample spectrum to the spectral window.</li>
		<li>Click on Files &#62; Save &#62; Save As and enter the file name followed by extension .spa and click on OK.</li>
	</ol>
	</li>
</ol>
7. Biosurfactant application (enhanced oil recovery)
NOTE: In this experiment, double distilled water was used as a negative control and 10% SDS, 10% Tween 80, and 10% commercial saponin were used as positive controls.
<ol>
	<li>Take the glass and seal the bottom outlet with glass wool and glass beads.</li>
	<li>Pack the column with sandy soil in such a way that some liquid can be added at the top of the soil and the flow through can be collected at the bottom. Mount the column on the holder and add some glass beads on top of the soil.</li>
	<li>Flood the column with 50 mL of brine solution and collect the flow through to determine the pore volume. 
	pore volume = volume of brine added on top - volume of flowthrough collected.</li>
	<li>Remove the brine from the column by forcing crude oil to pass through it after adding from the top of the column. Collect the volume of the brine and oil coming out of the column to determine initial oil saturation volume. The volume of the brine released from the column will be the initial oil saturation volume or original oil in place.</li>
	<li>Leave the column undisturbed for 24 h.</li>
	<li>After 24 h, flood the column with 10 pore 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.</li>
	<li>Prepare a mixture of biosurfactants by adding equal volumes of the extracted biosurfactant (extracted after step 3.5) to the glass beaker. Add the biosurfactants to the top of the column and incubate the column for 24 h.</li>
	<li>After 24 h, measure the amount of oil and water to determine additional or enhanced oil recovery. The volume of the oil released from the column will correspond to the residual oil recovered.</li>
	<li>Estimate enhanced oil recovery with the following equation: 
	<img alt="Equation 2" src="/files/ftp_upload/63207/63207eq02.jpg" style="margin: auto;" /></li>
</ol>

The authors declare no conflicts of interest.

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 capable of biosurfactant production. Here, we illustrate the methods for screening, identification, and application of a mixture of biosurfactants produced by Rhodococcus sp. IITD 102, Lysinibacillus sp. IITD 104, and Paenibacillus sp. IITD 108, for enhanced oil recovery. Biosurfactant production was screened by drop collapse, oil spreading, emulsion index assay and confirmed by measuring the reduction in surface tension of the medium due to microbial growth. Various reports on the production of glycolipid biosurfactants (rhamnolipids and trehalolipids) from Rhodococcus are available in literature19,20,21,22,23,24. Najafi et al. have reported production and optimization of a lipopeptide biosurfactant from Paenibacillus sp. alvei ARN6325. Bezza et al. have reported biodegradation of pyrene by a lipopeptide biosurfactant produced by Paenibacillus dendritiformis CN526. Biosurfactant production (Lipopeptides and glycolipids) has also been reported by other strains of Paenibacillus27,28,29,30,31. Various species of Lysinibacillus have been reported to produce biosurfactants32,33,34. Lysinibacillus sphaericus has been reported to produce rhamnolipid capable of solubilization of hydrophobic pesticides35.
One of the advantages that biosurfactants offer over their chemical counterparts is their stability under extreme environmental conditions. The biosurfactants produced by Rhodococcus sp. IITD 102, Lysinibacillus sp. IITD 104, and Paenibacillus sp. IITD 108 were assayed for their stability under different ranges of temperature, pH, and salt concentrations and were found to be stable under extreme values of these parameters. Previously, Habib et al. reported a lipopeptide produced by hydrocarbon degrading Rhodococcus sp. that showed stability across different ranges of temperatures, pH values, and salt concentrations36. Increase in concentration of inorganic salts has been reported to increase the stability of the emulsion37. The tendency of the colloids to agglomerate or separate is a function of the attractive (Van der Walls forces) and repulsive forces (electrostatic forces) that are involved during particle interaction38. The salt crystals on dissolving into water establish their own electrical charges and the ions released adsorb onto the emulsion droplets. On increasing the salt concentration, the expansion and repulsion of the second layer reduces. Also, the higher the charge density of an ion, the lower is the length of the electrical layer. Thus, divalent cations such as Na2+ result in the formation of more stable emulsions in comparison to monovalent cations39.
Another advantage that biosurfactants have over chemical surfactants is that they are biodegradable40. Zeng et al. have compared the degradation capacities of synthetic surfactant Triton X 100, linear alkylbenzene sulphonates (LAS) and rhamnolipid and found that the rhamnolipid biosurfactant was completely degraded whereas LAS and Triton X 100 were only partially degraded41. Liu et al. also report that in contrast to synthetic surfactants CTAB, Triton X 100 and SDS, rhamnolipid exhibit no toxicity and could be degraded easily by B. subtilis and other compost microorganisms42.
The biosurfactants produced by all three microbial strains were found to be glycolipids. Rhodococcus has previously been reported to produce glycolipid by various research groups20,21,23. Similar reports on glycolipid biosurfactant production by Lysinibacillus and Paenibacillus are also available in the literature31,32,35,43,44. Chemical identification of the biosurfactants revealed them to be rhamnolipids. Rhamnolipids are the class of glycolipid biosurfactants that contain one or two rhamnose units connected to a lipid chain45. They are the most studied type of biosurfactants. Various microbial strains have been reported to produce rhamnolipids7,46,47,48,49. Rhamnolipids have been reported to exhibit high potential for enhancing the recovery of residual oil50,51,52,53. In our study, we found that the mixture of biosurfactants produced by Rhodococcus sp. IITD 102, Lysinibacillus sp. IITD 104, and Paenibacillus sp. IITD 108 successfully recovered around 56.52% of the residual oil in a simulated sand pack column test. This showed that the mixture of biosurfactants can be used for the recovery of residual oil from the ground reservoirs. In a similar sand pack test, Sun et al. have reported that biosurfactant was successful in recovering 50% of the residual oil54. Biosurfactants containing cell-free broth of Bacillus subtilis have also been reported to be effective in recovering 33% of residual oil55. Residual oil recovery of 27% and 26%-36% have also been reported by Darvishi et al. and Wahabi et al.56,57.
Economic evaluation of biosurfactants for recovery of residual oil from reservoirs show that utilization of biosurfactants in EOR is economically a viable option. Moutinho et al. reported that the typical cost of a commercial biosurfactant (rhamnolipids) is around 59.6 USD per kg58. In another study, it has also been reported that the biosurfactant concentration of 28 mg/L of biosurfactant enhanced the residual oil recovery and led to the production of 52.5 m3 of additional oil59. The studies showed that biosurfactant concentration of 10 mg/L was sufficient to mobilize the oil recovery. According to the reported data, the amount of biosurfactant required to produce 52.5 m3 of additional oil is around 0.525 kg. The overall cost of production of 52.5 m3 oil is about US$ 21463 out of which only US$30 is the cost of production of the biosurfactant. The data shows that the percentage cost of the biosurfactant in oil production per barrel is only 0.0000139%.
Our results suggest that the combination of the biosurfactants can efficiently be used to recover residual oil from the reservoirs. To our knowledge, this is the first report on enhancing the recovery of the residual oil from the reservoir using a mixture of biosurfactants produced by different microbial strains. Although our study clearly describes the methods involved in screening, structural characterization, and application of biosurfactants in enhanced oil recovery, the study does not provide a detailed functional characterization of the biosurfactants, which affect their efficiency in various applications. Critical micelle concentration, which is the measure of the efficiency of any surfactant in forming the micelles and specifies the limiting concentration of the surfactant for its meaningful use, has not been determined in the present study60. Similarly, thermal stability of the biosurfactant, which determines its applicability at reservoir conditions for EOR has also not been described61. Biosurfactants in some applications are also used as antibiofilm agents. Their surface wettability plays an important role in determining their antibiofilm nature. Surface wettability studies have also not been carried out in the present work62. Other functional characteristics important in various applications of biosurfactants, which include their biodegradability and the antimicrobial nature have also not been determined in this study63,64. Thus, we have focused on the structural characterization of biosurfactants. Depending upon the target application, functional characterization such as stability, biodegradability, and antimicrobial activity may be performed.
In the drop collapse assay and oil spreading assay, to increase the visibility, it is better to use an oil that has some color. In the oil spreading assay, the emulsion should be observed after 24 h. Light foams, if formed, disintegrate in 24 h. Tensiometers are very sensitive instruments therefore during surface tension measurements, the vessel and the probe should be cleaned properly before every measurement to avoid any errors due to carry overs of last measurements. Extraction of biosurfactant involves the addition of chloroform methanol mixture to the cell-free supernatant. The step should either be performed in a fume hood, or the flask should be covered with aluminum foil immediately after transfer of the extraction mixture. During secondary recovery in an EOR experiment, brine solution should be added in excess until no further oil comes out of the column.
The method discusses the scope of mixture of biosurfactants in the recovery of residual oil from the columns. The process is dependent on many factors. The growth stage of the microbes at which the initial cultures are harvested. Some biosurfactants have been reported to be produced in the log phase, while others have been reported to be produced in the stationary phase. The cultures should be harvested accordingly at the particular stage when biosurfactant production has reached its maximum. Biosurfactant screening assays are less sensitive, therefore all the assays should be performed before reaching a conclusion about the biosurfactant producing ability of a particular strain. Purification of the biosurfactant should be performed before chemical characterization of the biosurfactant if the concentration of biosurfactant is low in the crude biosurfactant. Enhanced oil recovery experiments are highly dependent on the type of soil used for packing the column. The soil must be completely dry and should be sieved to remove larger granules and other solid contaminants. A mixture of sandy soil and dry garden soil (in equal ratio) should be preferred for packing the column. The column outlet must be sealed properly with glass wool and glass beads to avoid leakage of soil from the column during the course of the experiment.
The method described is useful for determining the significance of the produced biosurfactants and their mixtures in recovery of additional oil in a simulated sand pack column experiment. Different biosurfactants have different specificities because they contain different functional groups65. A combination of biosurfactants will enable solubilization of diverse hydrocarbons and will therefore increase the residual oil recovery from the reservoirs. The method described will help in determining the potential of biosurfactant mixtures in field applications such as enhanced oil recovery from oil wells.

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 other1,3. 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 &#176;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63207/63207fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 strains15,16. 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.

<table><tbody><tr><td>1 ml pipette</td><td>Eppendorf, Germany</td><td>G54412G</td><td></td></tr><tr><td>&lt;sup&gt;1&lt;/sup&gt;H NMR</td><td>Bruker Avance AV-III type spectrometer,USA</td><td></td><td></td></tr><tr><td>20 ul pipette</td><td>Thermo scientific, USA</td><td>H69820</td><td></td></tr><tr><td>Autoclave</td><td>JAISBO, India</td><td>Ser no 5923</td><td>Jain Scientific</td></tr><tr><td>Blue flame burner</td><td>Rocker scientific, Taiwan</td><td>dragon 200</td><td></td></tr><tr><td>Butanol</td><td>GLR inovations, India</td><td>GLR09.022930</td><td></td></tr><tr><td>C18 column</td><td>Agilent Technologies, USA</td><td>770995-902</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Germany</td><td>5810R</td><td></td></tr><tr><td>Chloroform</td><td>Merck, India</td><td>1.94506.2521</td><td></td></tr><tr><td>Chloroform-d</td><td>SRL, India</td><td>57034</td><td></td></tr><tr><td>Falcon tubes</td><td>Tarsons, India</td><td>546041</td><td>Radiation sterilized polypropylene</td></tr><tr><td>FT-IR</td><td>Thermo Fisher Scientific, USA</td><td>&amp;nbsp;Nicolet iS50</td><td></td></tr><tr><td>Fume hood</td><td>Khera, India</td><td>47408</td><td>Customied</td></tr><tr><td>glacial acetic acid</td><td>Merck, India</td><td>1.93002</td><td></td></tr><tr><td>Glass beads</td><td>Merck, India</td><td>104014</td><td></td></tr><tr><td>Glass slides</td><td>Polar industrial Corporation, USA</td><td>Blue Star</td><td>75 mm * 25 mm</td></tr><tr><td>Glass wool</td><td>Merk, India</td><td>104086</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck, India</td><td>1003170510</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific, USA</td><td>MaxQ600</td><td>Shaking incubator</td></tr><tr><td>Incubator</td><td>Khera, India</td><td>Sunbim</td><td></td></tr><tr><td>Iodine resublimed</td><td>Merck, India</td><td>231-442-4</td><td>&amp;nbsp;resublimed Granules</td></tr><tr><td>K12 &amp;ndash;Kruss tensiometer</td><td>Kruss Scientific, Germany</td><td>K100</td><td></td></tr><tr><td>Laminar air flow cabnet</td><td>Thermo Scientific, China</td><td>1300 Series A2</td><td></td></tr><tr><td>LCMS</td><td>Agilent Technologies, USA</td><td>1260 Infinity II</td><td></td></tr><tr><td>Luria Broth</td><td>HIMEDIA, India</td><td>M575-500G</td><td>Powder</td></tr><tr><td>Methanol</td><td>Merck, India</td><td>107018</td><td></td></tr><tr><td>Ninhydrin</td><td>Titan Biotech Limited, India</td><td>1608</td><td></td></tr><tr><td>p- anisaldehyde</td><td>Sigma, USA</td><td>204-602-6</td><td></td></tr><tr><td>Petri plate</td><td>Tarsons, India</td><td>460090-90 MM</td><td>Radiation sterilized polypropylene</td></tr><tr><td>Saponin</td><td>Merck, India</td><td>232-462-6</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck, India</td><td>231-598-3</td><td></td></tr><tr><td>Test tubes</td><td>Borosil, India</td><td>9800U06</td><td>Glass tubes</td></tr><tr><td>TLC plates</td><td>Merck, India</td><td>1055540007</td><td></td></tr><tr><td>Vortex</td><td>GeNei, India</td><td>2006114318</td><td></td></tr><tr><td>Water Bath</td><td>Julabo, India</td><td>SW21C</td><td></td></tr></tbody></table>

enhanced oil recovery using a combination of biosurfactants

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.

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 (Figure 4a). On the other hand, the droplet of water did not collapse and was therefore scored negative for the presence of the biosurfactant. Commercial surfactant and the cell free supernatants of the three bacterial cultures were also successful in displacing the layer of oil in the oil spreading assay and, therefore, were scored positive for the presence of biosurfactant (Figure 4b). Water, on the other hand, could not displace any oil and, therefore, was scored negative. In emulsion index assays, a stable emulsion was observed in test tubes containing commercial surfactant and the supernatants of the three microbial strains. However, no emulsion was observed in the test tube containing uninoculated culture medium (Figure 4c). This again suggested that Rhodococcus sp. IITD102, Lysinibacillus sp. IITD104, and Paenibacillus sp. IITD108 produce biosurfactants. Confirmation of biosurfactant production was obtained by measuring the surface tension of the cell-free broth and comparing it with the uninoculated control. The biosurfactants of Rhodococcus sp. IITD102, Lysinibacillus sp. IITD104, and Paenibacillus sp. IITD108 were found to reduce the surface tension of the medium from 58.89 mN/m to 45.41 mN/m, 45.82 mN/m, and 28.43 mN/m, respectively (Figure 5).
IFT measurements were performed using the ring pull method. The biosurfactants from all the three strains were capable of significantly reducing the interfacial tensions between various aqueous and organic phases (Table S1). The surface tension and the interfacial tension measurements confirm that all the three strains produce biosurfactants.
Solvent extraction of biosurfactants from the cell free cultures of Rhodococcus sp. IITD102, Lysinibacillus sp. IITD104, and Paenibacillus sp. IITD108 resulted in biosurfactant concentrations of 820 mg/L, 560 mg/L, and 480 mg/L, respectively.
As observed in Figure 4C, the emulsion formed by the biosurfactant was water in oil microemulsion. Emulsion stability assays showed that the biosurfactants exhibited a good stability under diverse environmental conditions&#160;(Figure 6). The emulsions produced were very stable across diverse temperatures (Figure 6a), pH values (Figure 6b), and salt concentrations (Figure 6c) tested.
Thin Layer Chromatography was performed to determine the nature of the biosurfactants. Staining the plates with iodine vapor resulted in the development of yellow spots in all the biosurfactants and the control (Biosurfactant from Bacillus sp. IITD 106 (Figure 7a). This indicated that the biosurfactants contained a lipid moiety. No blue colored spots were obtained in any of the TLC plates upon staining with ninhydrin (Figure 7b). This showed that the biosurfactants did not contain any peptide moiety. Blue and green spots were observed in all the TLC plates, when stained with anisaldehyde stain (Figure 7c). This showed that the biosurfactants contained a carbohydrate moiety. From the results of TLC, it was concluded that all the biosurfactants were glycolipids.
Chemical identification of the biosurfactants using LCMS revealed that the Rhodococcus sp. IITD102, and Lysinibacillus sp. IITD104 produce the same type of biosurfactant, that was identified as Di-rhamnopyranosic hydroxydecanoic acid with a mass of 480.25 Da. The structure of biosurfactant was supported by 1H NMR and FT-IR data (Figure 8).
In 1H NMR spectrum, the chemical shifts obtained at 7.2 represented the protons of carboxylic groups. Chemical shifts corresponding to protons of methyl group were obtained in the range 1-2 ppm. Shifts corresponding to protons attached to alkyl groups were obtained at 2.3 ppm. FT-IR spectra of the biosurfactants extracted from Rhodococcus sp. IITD102 and Lysinibacillus sp. IITD104 showed the presence of strong peaks at wave number 3,290 cm-1, which confirms the presence of the OH functional group. A small peak at wave number 2,951 cm-1 corresponds to CH stretching. A strong peak at 1,620 cm-1 represented the presence of carboxylic group in the biosurfactants. Other peaks that were obtained at 1,530, 1,410, 1,200, and 1,060 confirmed the presence of alkyl, CH3, C-O-C, and C-CH3 functional groups, respectively. Both NMR and FT-IR data supported the structure of the biosurfactant determined from LCMS studies. LCMS of crude biosurfactant from Paenibacillus sp. IITD108 (Figure 9) showed that it produces a rhamnolipid containing three lipid chains forming the bulk hydrophobic core of the biosurfactant. The biosurfactant was identified as 2decanoyl)-&#945;L rhamnopyranosyl-3-hydroxydecanoic acid with a mass of 802 Da. The results of LCMS were supported by 1H NMR and FT-IR data.
The set up for enhanced oil recovery is shown in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63207/63207fig2v2.jpg" /> 
Figure 2: Experimental set up for enhanced oil recovery using the sand pack column technique. The column packed with soil was mounted on the holder. The bottom outlet was sealed with glass wool and glass beads. After secondary recovery, the residual oil inside the column was subjected to enhanced oil recovery by addition of the biosurfactant mixture to it. The tube placed at the bottom of the column was used to collect the eluted fraction. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the simulated enhanced oil recovery experiment, out of 50 mL of brine added to the top of the column, 12 mL was collected in the flowthrough and, therefore, the pore volume was estimated to be 38 mL. When oil was forced through the column, 33 mL of brine was released from the column. This represented initial oil saturation volume. Secondary recovery using 10 pore volumes of brine resulted in the elution of 10 mL of oil. The residual oil left in the column was 23 mL. Water containing the mixture of biosurfactants was able to recover 13 mL of oil from the column (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63207/63207fig3v2.jpg" /> 
Figure 3: Simulated enhanced oil recovery using a sand pack column. The initial oil saturation volume of both control and test column was around 33 mL. During secondary oil recovery, around 10 mL of the oil was recovered from both control and the test columns. The differences in the recovery profiles of the test and the control columns were observed only during recovery of the residual oil left in the column. The biosurfactant mixture resulted in further recovery of 13 mL of the residual oil left from the test column while in the control column only 1.03 mL of oil was recovered in this step. This shows that the biosurfactant mixture has great potential in enhancing the recovery of residual oil from the reservoirs. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This represented enhanced oil recovery. Therefore, water containing the mixture of biosurfactants was capable of recovering 56.52% of the residual oil from the column (Table 1). On the other hand, solutions of 10% SDS, 10% Tween 80, and 10% saponin were able to recover 85%, 68%, and 73% residual oil from the column.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td>Control flooding</td>
			<td>Combined Biosurfactant flooding</td>
			<td>10 % SDS</td>
			<td>10 % Tween</td>
			<td>10 % Saponin</td>
		</tr>
		<tr>
			<td>PV (mL)</td>
			<td>37</td>
			<td>38</td>
			<td>38</td>
			<td>35</td>
			<td>37</td>
		</tr>
		<tr>
			<td>OOIP / IOSV (mL)</td>
			<td>33</td>
			<td>33</td>
			<td>33</td>
			<td>29</td>
			<td>33</td>
		</tr>
		<tr>
			<td>POS (%)</td>
			<td>89.91</td>
			<td>86.84</td>
			<td>86.84</td>
			<td>82.85</td>
			<td>89.18</td>
		</tr>
		<tr>
			<td>SV (ml)</td>
			<td>330</td>
			<td>330</td>
			<td>330</td>
			<td>330</td>
			<td>330</td>
		</tr>
		<tr>
			<td>SOR (mL)</td>
			<td>9.77</td>
			<td>10</td>
			<td>11.5</td>
			<td>9.2</td>
			<td>10</td>
		</tr>
		<tr>
			<td>ROC (mL)</td>
			<td>23.23</td>
			<td>23</td>
			<td>21.5</td>
			<td>18.8</td>
			<td>23</td>
		</tr>
		<tr>
			<td>ROS (%)</td>
			<td>70.39</td>
			<td>69.69</td>
			<td>65.15</td>
			<td>64.82</td>
			<td>69.69</td>
		</tr>
		<tr>
			<td>Rv (ml)</td>
			<td>60</td>
			<td>60</td>
			<td>60</td>
			<td>60</td>
			<td>60</td>
		</tr>
		<tr>
			<td>ROR (mL)</td>
			<td>1.03</td>
			<td>13</td>
			<td>18.5</td>
			<td>12.8</td>
			<td>16.8</td>
		</tr>
		<tr>
			<td>AOR (%)</td>
			<td>4.43</td>
			<td>56.5</td>
			<td>85</td>
			<td>68.08</td>
			<td>73.04</td>
		</tr>
	</tbody>
</table>
where PV = pore volume determined after initial column saturation with brine, OOIP = original oil in place, IOSV = initial oil saturation volume, POS = Percentage oil saturation, Sv = volume of brine added for secondary recovery, SOR = secondary oil recovered after brine flooding, ROC = residual oil in column after secondary recovery, ROS = residual oil saturation, ROR = Residual oil recovered after biosurfactant flooding, AOR = additional oil recovered&#160;
Table 1: Simulated enhanced oil recovery in a sand pack column.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63207/63207fig4v2.jpg" /> 
Figure 4: Screening assays for biosurfactant production (a) Drop collapse assay: The drop of water did not collapse after being added to the oil-coated surface while the chemical surfactant and cell-free supernatants of the three bacterial strains resulted in the drop collapse. (b) Oil displacement assay: The drop of water did not result in displacement of the oil while the chemical surfactant and cell-free supernatants of the three bacterial strains displaced the layers of oil (c) Emulsion index assay: The cell free supernatants and the commercial surfactant solution all resulted in the formation of a stable emulsion index. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63207/63207fig5v2.jpg" /> 
Figure 5: Surface tension reduction due to biosurfactant production.&#160;Determination of reduction in the surface tension of the medium due to microbial growth confirmed the biosurfactant production by the microbial members. Due to growth of Rhodococcus sp. IITD 102 and Lysinibacillus sp. IITD 104, the surface tension of the medium reduced from 59 mN/m to 45 mN/m. Due to growth of Paenibacillus sp. IITD 108, the surface tension of the medium reduced from 59 mN/m to 28 mN/m. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63207/63207fig6v2.jpg" /> 
Figure 6: Emulsion stability at various (a) temperatures, (b) pH values and (c) salt concentrations. All the emulsions generated using the supernatants of the three microbial strains and the mixture of commercial surfactant show great stability under different environmental conditions. Within the ranges of temperature, pH, and salt concentration tested, the emulsion indices determined were similar and no major reduction of EI was observed at higher values of the varying factors implying that biosurfactant can be used under extreme environmental conditions. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63207/63207fig7v2.jpg" /> 
Figure 7: TLC characterization of biosurfactants&#160;(a) Plates stained with iodine vapor: Various spots developed on the TLC plate show that the extracted biosurfactants are a mixture of various compounds containing a lipid group. The spots marked with blue arrow represent the biosurfactants, which have stained positive for the presence of lipid moiety. The other spots represent the rest of the compounds present in the mixture of the crude biosurfactant. (b) Plates stained with ninhydrin: No purple spots appeared when the plates were stained with ninhydrin. This represented absence of any amino acids in the biosurfactant mixture and (c) Plates stained with anisaldehyde: Light green and yellow spots appeared on the TLC plate and these represent the compounds containing sugars. The spots marked with black arrows represent the biosurfactants which have stained positive for the presence of the carbohydrate moiety. The spots which stained both with iodine and anisaldehyde represent compounds containing both lipid and carbohydrate moieties and could possibly be a glycolipid biosurfactant. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63207/63207fig8v2.jpg" /> 
Figure 8: Chemical characterization of the biosurfactant extracted from Rhodococcus sp. IITD 102 and Lysinibacillus sp. IITD 104. (a) represents the FT-IR spectra of the biosurfactants extracted from Rhodococcus sp. IITD 102 and Lysinibacillus sp. IITD 104, (b) represents the H1 NMR spectra of the biosurfactants extracted from Rhodococcus sp. IITD 102 and Lysinibacillus sp. IITD 104, and (c) shows the structure of the crude biosurfactants extracted from Rhodococcus sp. IITD 102 and Lysinibacillus sp. IITD 104. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63207/63207fig9v2.jpg" /> 
Figure 9: Chemical characterization of the biosurfactant extracted from Paenibacillus sp. IITD 108. (a) represents the FT-IR spectra of the biosurfactants extracted from Paenibacillus sp. IITD 108, (b) represents the 1H NMR spectra of the biosurfactants extracted from Paenibacillus sp. IITD 108, and (c) shows the structure of the crude biosurfactants extracted from Paenibacillus sp. IITD 108. <a href="https://www.jove.com/files/ftp_upload/63207/63207fig9v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure S1: Structure of different types of biosurfactants. <a href="https://www.jove.com/files/ftp_upload/63207/supplementary information2.pdf" target="_blank">Please click here to download this File.</a>
Table S1: Effect of biosurfactants on the interfacial tension (IFT) between water and hydrocarbons. <a href="https://www.jove.com/files/ftp_upload/63207/Table S1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Enhanced Oil Recovery using a Combination of Biosurfactants at JoVE.com

Enhanced Oil Recovery using a Combination of Biosurfactants

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&#160;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 ...

enhanced-oil-recovery-using-a-combination-of-biosurfactants

Universidad Científica del Sur

Research

JoVE Journal

Environment

5.1K Views.  Indian Institute of Technology Delhi. 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.

Video: Enhanced Oil Recovery using a Combination of Biosurfactants

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Bioengineering

Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are resistant to low molecular-weight oil components, unlike traditional polydimethylsiloxane (PDMS) devices. Here, we demonstrate a facile method for making a device with this property, and we use the product of this protocol for examining the pore-scale mechanisms by which foam recovers crude oil. A pattern is first designed using computer-aided design (CAD) software and printed on a transparency with a high-resolution printer. This pattern is then transferred to a photoresist via a lithography procedure. PDMS is cast on the pattern, cured in an oven, and removed to obtain a mold. A thiol-ene crosslinking polymer, commonly used as an optical adhesive (OA), is then poured onto the mold and cured under UV light. The PDMS mold is peeled away from the optical adhesive cast. A glass substrate is then prepared, and the two halves of the device are bonded together. Optical adhesive-based devices are more robust than traditional PDMS microfluidic devices. The epoxy structure is resistant to swelling by many organic solvents, which opens new possibilities for experiments involving light organic liquids. Additionally, the surface wettability behavior of these devices is more stable than that of PDMS. The construction of optical adhesive microfluidic devices is simple, yet requires incrementally more effort than the making of PDMS-based devices. Also, though optical adhesive devices are stable in organic liquids, they may exhibit reduced bond-strength after a long time. Optical adhesive microfluidic devices can be made in geometries that act as 2-D micromodels for porous media. These devices are applied in the study of oil displacement to improve our understanding of the pore-scale mechanisms involved in enhanced oil recovery and aquifer remediation.

The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported.

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Chemistry

Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography

In situ wettability measurements in hydrocarbon reservoir rocks have only been possible recently. The purpose of this work is to present a protocol to characterize the complex wetting conditions of hydrocarbon reservoir rock using pore-scale three-dimensional X-ray imaging at subsurface conditions. In this work, heterogeneous carbonate reservoir rocks, extracted from a very large producing oil field, have been used to demonstrate the protocol. The rocks are saturated with brine and oil and aged over three weeks at subsurface conditions to replicate the wettability conditions that typically exist in hydrocarbon reservoirs (known as mixed-wettability). After the brine injection, high-resolution three-dimensional images (2 &#181;m/voxel) are acquired and then processed and segmented. To calculate the distribution of the contact angle, which defines the wettability, the following steps are performed. First, fluid-fluid and fluid-rock surfaces are meshed. The surfaces are smoothed to remove voxel artefacts, and in situ contact angles are measured at the three-phase contact line throughout the whole image. The main advantage of this method is its ability to characterize in situ wettability accounting for pore-scale rock properties, such as rock surface roughness, rock chemical composition, and pore size. The in situ wettability is determined rapidly at hundreds of thousands of points. 
The method is limited by the segmentation accuracy and X-ray image resolution. This protocol could be used to characterize the wettability of other complex rocks saturated with different fluids and at different conditions for a variety of applications. For example, it could help in determining the optimal wettability that could yield an extra oil recovery (i.e., designing brine salinity accordingly to obtain higher oil recovery) and to find the most efficient wetting conditions to trap more CO2 in subsurface formations.

This protocol is presented to characterize the complex wetting conditions of an opaque porous medium (hydrocarbon reservoir rock) using three-dimensional images obtained by X-ray microtomography at subsurface conditions.

In situ wettability measurements in hydrocarbon reservoir rocks have only been possible recently. The purpose of this work is to present a protocol to ...

Engineering

Studying Surfactant Effects on Hydrate Crystallization at Oil-Water Interfaces Using a Low-Cost Integrated Modular Peltier Device

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a temperature regulator, visualization techniques, and inner pressure measurements. The temperature control system contains a low-cost, programmable temperature regulator made with solid-state Peltier components. Along with the temperature control system, we incorporated visualization techniques and internal pressure measurements to study hydrate formation and inhibition in the presence of nonionic surfactants. We studied the hydrate-inhibiting ability of nonionic surfactants (sorbitane monolaurate, sorbitane monooleate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate) at low (i.e., 0.1 CMC), medium (i.e., CMC), and high (i.e., 10 CMC) concentrations. Two types of crystals were formed: planar and conical. Planar crystals were formed in plain water and low surfactant concentrations. Conical crystals were formed in high surfactant concentrations. The results of the study show that conical crystals are the most effective in terms of hydrate inhibition. Because conical crystals cannot grow past a certain size, the hydrate growth rate as a conical crystal is slower than the hydrate growth rate as planar crystal. Hence, surfactants that force hydrates to form conical crystals are the most efficient. The goal of the protocol is to provide a detailed description of an experimental system that is capable of investigating the cyclopentane hydrate crystallization process on the surface of a water droplet in the presence of surfactant molecules.

We present a protocol to study the formation of hydrates in the presence of nonionic surfactants on the interface of a water droplet submerged in cyclopentane. The protocol consists of building a low-cost, programmable, temperature regulator. The temperature control system is combined with visualization techniques and internal pressure measurements.

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a ...

Biodiesel Synthesis by Ultrasonic-Assisted Transesterification of Vegetable Oils

Ultrasonic-Assisted Preparation of Biodiesel Products from Vegetable Oils

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel synthesis. This alkaline-catalyzed procedure harnesses ultrasound as a potent energy input, facilitating the rapid conversion of extra virgin olive oil into biodiesel. In this demonstration, the reaction is run in an ultrasonic bath under ambient conditions for 15 min, requiring a 1:6 molar ratio of extra virgin olive oil to methanol and a minimum amount of KOH as the catalyst. The physiochemical properties of biodiesel are also reported. Emphasizing the remarkable advantages of ultrasonic-assisted transesterification, this method demonstrates notable reductions in reaction and separation times, achieving near-perfect purity (~100%), high yields, and negligible waste generation. Importantly, these benefits are achieved within a framework that prioritizes safety and environmental sustainability. These compelling findings underscore the effectiveness of this approach in converting vegetable oil into biodiesel, positioning it as a viable option for both research and practical applications.

Author Spotlight: Employing Green-Chemistry Principles for Safe and Sustainable Synthesis of Biodiesels

A safe ultrasonic-assisted transesterification method for vegetable oils using an alkaline catalyst is presented here. The method is rapid and efficient for preparing pure biodiesel products.

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Biology

Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; however, they can disturb the lipid profile of the patients. In this study, self-nanoemulsifying drug delivery systems of omega-3 fatty acids-rich oils are prepared and optimized for the delivery of lipophilic drugs. Rosuvastatin, a potent hypolipidemic drug, was used as a model lipophilic drug. Fish oil showed more than 7-fold higher solubility of rosuvastatin than other oils and therefore it was selected for the development of self-nanoemulsifying drug delivery systems (SNEDDS). Different combinations of surfactants and co-surfactants were screened and a surfactant mixture of Tween 80 (surfactant) and Capryol PGMC (cosurfactant) were selected for compatibility with fish oil and rosuvastatin. A pseudoternary phase diagram of oil, surfactant, and co-surfactant was designed to identify the emulsion region. The pseudoternary phase diagram predicted a 1:3 oil and surfactant mixture as the most stable ratio for the emulsion system. Then, a response-surface methodology (Box-Behnken design) was applied to calculate the optimal composition. After 17 runs, fish oil, Tween 80, and Capryol PGMC in proportions of 0.399, 0.67, and 0.17, respectively, were selected as the optimized formulation. The self-nanoemulsifying drug delivery systems showed excellent emulsification potential, robustness, stability, and drug release characteristics. In the drug release studies, SNEDDS released 100% of the payload in around 6 h whereas, release of the plain drug was less than 70% even after 12 h. Therefore, omega-3 fatty acids-rich healthy lipids have enormous potential to enhance the solubility of lipophilic drugs whereas, self-emulsification can be used as a simple and feasible approach to exploit this potential.

Oils used for drug delivery applications can disrupt the lipid profile of patients, which is undesirable in cardiovascular diseases. Omega-3 fatty acids-rich oils are a healthy alternative to conventional oils and have enormous potential for self-emulsified drug delivery systems.

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; ...