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://jove.unicartagenaproxy.elogim.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 (<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 ...

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Environment

5.0K 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

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

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

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. ...

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; ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...