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 capab...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml pipette</td>
			<td>Eppendorf, Germany</td>
			<td>G54412G</td>
			<td></td>
		</tr>
		<tr>
			<td>1H 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>&#160;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>&#160;resublimed Granules</td>
		</tr>
		<tr>
			<td>K12 &#8211;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 (<stron...

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

Microsoft Bing

Research

JoVE Journal

Environment

4.9K 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

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Chemical Analysis of Water-accommodated Fractions of Crude Oil Spills Using TIMS-FT-ICR MS

Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system requires the careful preparation of the sample in order to accurately replicate the natural formation of the water-accommodated fraction that occurs in nature. Low-energy water-accommodated fractions (LEWAF) are carefully prepared by mixing crude oil and water at a set ratio. Aspirator bottles are then irradiated, and at set time points, the water is sampled and extracted using standard techniques. A second challenge is the representative characterization of the sample, which must take into consideration the chemical changes that occur over time. A targeted analysis of the aromatic fraction of the LEWAF can be performed using an atmospheric-pressure laser ionization source coupled to a custom-built trapped ion mobility spectrometry&#8211;Fourier transform&#8211;ion cyclotron resonance mass spectrometer (TIMS-FT-ICR MS). The TIMS-FT-ICR MS analysis provides high-resolution ion mobility and ultrahigh-resolution MS analysis, which further allow the identification of isomeric components by their collision cross-sections (CCS) and chemical formula. Results show that as the oil-water mixture is exposed to light, there is significant photo-solubilization of the surface oil into the water. Over time, the chemical transformation of the solubilized molecules takes place, with a decrease in the number of identifications of nitrogen- and sulfur-bearing species in favor of those with a greater oxygen content than were typically observed in the base oil.

The low-energy water-accommodated fraction (LEWAF) of crude oil is a challenging system to analyze, because over time, this complex mixture undergoes chemical transformations. This protocol illustrates methods for the preparation of the LEWAF sample and for performing photo-irradiation and chemical analysis by trapped ion mobility spectrometry&#8211;FT-ICR MS.

Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

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 ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...