We acknowledge the financial support from the Rice University Consortium for Processes in Porous Media (Houston, TX, USA).

Caution: This protocol involves handling a high temperature oven, toxic chemicals, and UV light. Please read all the material safety data sheets carefully and follow your institution&#39;s chemical safety guidelines.
1. Device Design
<ol>
	<li>Design a photomask in a CAD software application.
	<ol>
		<li>Draw a rectangular channel that is 3 cm long and 0.5 cm wide (Figure 1b-top right).</li>
		<li>Create an array of enclosed shapes representing the grains of the porous media. 
		NOTE: These shapes are referred to as posts because they will become three-dimensional structures during the soft lithography process. The shape and size of the posts should be on the order of tens of microns, and have a spacing of ten to one hundred microns. Multiple post sizes may be employed to create heterogeneity, and a section can be left bare of posts to simulate a fracture in the media.</li>
		<li>Draw inlet and outlet channels that are approximately one-third as wide as the porous media section. Draw a channel stemming from the inlet port to act as a drain.</li>
		<li>Draw a bounding box around the entire design with a minimum of 1.0 cm of clearance from the design. 
		NOTE: The area between the bounding box and the borders of the design, as well as the posts, are to be made transparent on the photomask.</li>
	</ol>
	</li>
	<li>Submit the CAD file to a company for high-resolution CAD printing 
	NOTE: Optional: For a foam displacement experiment, design a microfluidic foam generator (Figure 1a). Repeat step 1, omitting the design heterogeneity and bounding box. A flow-focusing geometry is recommended at the inlet before the porous media design. The flow spaces should be transparent on the photomask.</li>
</ol>
2. PDMS Mold Fabrication
<ol>
	<li>Create a photoresist-patterned silicon wafer master mold in a clean room
	<ol>
		<li>Spin-coat a 20 &#181;m layer of photoresist onto a new silicon wafer at 2,000 rpm for 30 s.</li>
		<li>Soft bake the wafer on a hot plate in two increments: 65 &#176;C for 1 min followed by 95 &#176;C for 3 min.</li>
		<li>Use a mask aligner to pattern the photoresist layer with the CAD design using a constant dosage of 150 mJ/cm2.</li>
		<li>Perform a post-exposure bake on a hot plate in two increments: 65 &#176;C for 1 min followed by 95 &#176;C for 3 min. Allow the wafer to cool for 5 min.</li>
		<li>Immerse the wafer in 100 mL of propylene-glycol-methyl-ether-acetate in a glass crystallization dish. Gently agitate by hand for 10 min to develop the photoresist pattern. Rinse it with isopropanol and dry the wafer under a stream of dry air.</li>
		<li>Hard bake the wafer on a hot plate in two increments: 120 &#176;C for 5 min followed by 150 &#176;C for 10 min. Allow the wafer to cool for 15 min.</li>
	</ol>
	</li>
	<li>Cast PDMS onto the silicon wafer master mold
	<ol>
		<li>Mix a total of 30 g of the PDMS elastomer and curing agent in a 5:1 ratio inside a dust-free disposable container.</li>
		<li>Degas the PDMS in a vacuum desiccator for 30 min.</li>
		<li>Pour the PDMS onto the photoresist-patterned silicon wafer master mold in a 150 mm glass Petri dish.</li>
		<li>Place the Petri dish containing the wafer and PDMS in an 80 &#176;C oven for 1 h.</li>
		<li>Remove the Petri dish from the oven and allow the contents to reach room temperature. 
		NOTE: The procedure may be paused at this point.</li>
	</ol>
	</li>
	<li>Prepare the PDMS mold for pattern transfer to optical adhesive
	<ol>
		<li>Carefully cut the PDMS mold out using a scalpel, and peel the mold away from the wafer.</li>
		<li>Clean and protect the PDMS mold using clear adhesive tape. 
		NOTE: The procedure may be paused at this point.</li>
		<li>Place the PDMS mold, pattern-side up, in the bottom of a dust-free 60 mm plastic Petri dish. Allow 10 s for the PDMS to stick to the plastic.</li>
		<li>Protect the surface of the PDMS with clear plastic tape until step 3.1.1. 
		NOTE: Optional: To make the foam generator, repeat steps 2.1. through 2.3.2. for the foam-generator design.</li>
	</ol>
	</li>
</ol>
3. Optical Adhesive Device Fabrication
<ol>
	<li>Cast optical adhesive onto the PDMS mold
	<ol>
		<li>Remove the tape from the patterned surface of the PDMS, and pour optical adhesive into the 150 mm Petri dish to a depth of approximately 0.9 cm above the top surface of the PDMS mold. Gently remove any bubbles with any type of cotton swab.</li>
	</ol>
	</li>
	<li>Cure the optical adhesive under UV light for a total of 40 min as outlined in steps 3.2.1 - 3.2.5 in a PSD-UV system. 
	Caution: Wear appropriate protection when working with UV light.
	<ol>
		<li>Expose the Petri dish to UV light (254 nm) for 5 min.</li>
		<li>Invert the Petri dish such that the bottom is now facing the UV source, and expose the under-side to UV light for 5 min.</li>
		<li>Invert the Petri dish, return it to the upright position, and re-expose the top side to UV light for 5 min.</li>
		<li>Invert the Petri dish upside down again, and re-expose the bottom side to UV light for 10 min.</li>
		<li>Invert the Petri dish back to the upright position, and re-expose the top side to UV light for 15 min. 
		NOTE: The curing procedure in steps 3.2.1 through 3.2.5 is only applicable when the specified PSD-UV apparatus is used (Table of Materials). Cure times will vary depending on the specific lamp that is used and on the exact thickness of the optical adhesive layer.</li>
	</ol>
	</li>
	<li>Remove the cured optical adhesive from the PDMS mold
	<ol>
		<li>Use a box cutter to carefully break the optical adhesive out of the Petri dish mold. 
		Caution: Box cutter blades are very sharp and can easily cut flesh. Be careful when working around the sharp edges of broken Petri dishes.</li>
		<li>Use a sturdy pair of scissors to remove excess optical adhesive from the edge of the design.</li>
		<li>Slowly peel the PDMS mold away from the optical adhesive puck. Protect the patterned portions of the optical adhesive surface and the PDMS surface with clear tape.</li>
		<li>Use a 1.0 mm biopsy punch to create inlet, outlet, and drain holes. Protect the patterned optical adhesive with clear tape.</li>
	</ol>
	</li>
	<li>Prepare the substrate
	<ol>
		<li>Dispense 1 mL of optical adhesive onto a new glass slide, and spin-coat the slide in two steps: 500 rpm for 5 s then 4,000 rpm for 20 s.</li>
		<li>Quickly transfer the substrate to the UV light treatment, and partially cure the thin optical adhesive layer under UV light for 30 s.</li>
	</ol>
	</li>
	<li>Bond the optical adhesive cast to the substrate
	<ol>
		<li>Place the optical adhesive cast, patterned side up, and the substrate, coated-side up, in an O2 plasma cleaner. Plasma clean the surface for 20 s at 540 mTorr.</li>
		<li>Firmly press the two treated surfaces together until all undesired air pockets have been minimized or removed.</li>
		<li>Fully cure the device under UV light for 20 min. 
		Caution: For UV light, wear appropriate protection such as protective glasses, lab coat, gloves, etc.</li>
		<li>Place the device on a hot plate at 50 &#176;C for 18 h.</li>
	</ol>
	</li>
	<li>Insert a 6-inch long segment of 0.58 mm ID low density polyethylene tubing (PE/3) into each of the ports on the device.</li>
	<li>Use a 5 min quick-set epoxy to secure the tubing in place. 
	NOTE: Optional: To complete the foam generator, repeat steps 3.5.1, 3.5.2, 3.6, and 3.7. using the foam-generator PDMS cast and a new glass slide, instead of the optical adhesive cast and prepared substrates, respectively.</li>
</ol>
4. Oil Displacement Experiment
<ol>
	<li>Prepare the microfluidic device to be imaged on an inverted microscope equipped with a high-speed camera. Fix the device to the microscope stage using tape. Using a 4X objective, focus on the area of interest (AOI).</li>
	<li>Prepare the injection fluids 
	NOTE: For three-phase systems, a dye should be added to clear displacing fluids to provide color contrast for image analysis.
	<ol>
		<li>Load 3 mL of crude oil or model oil sample into a 10 mL glass syringe equipped with a 23 gauge industrial dispensing tip. Secure the syringe in the syringe pump holder and set the appropriate diameter value on the syringe pump settings.</li>
		<li>Load 1 mL of the displacing fluid into a 3 mL plastic syringe equipped with a 23 gauge industrial dispensing tip. Secure the syringe in the syringe pump holder and set the appropriate diameter value on the syringe pump settings. 
		NOTE: Optional: For foam generation experiments, connect a 10 m long 25 &#181;m diameter glass capillary tube to an N2 gas tank and set the gas pressure to the desired value for the required gas flow rate as obtained from a calibration curve. Allow 10 min for the gas flow to equilibrate.</li>
	</ol>
	</li>
	<li>Saturate the OPTICAL ADHESIVE model porous media device with oil
	<ol>
		<li>Connect the displacing fluid to the inlet of the device by inserting the needle tip into the PE/3 tubing. 
		NOTE: Optional: When foam is used as the displacing phase, connect the displacing fluid syringe to the inlet of the foam generator. Connect the gas capillary to the second inlet port on the foam generator by inserting the capillary tube into a 23-gauge industrial dispensing tip and sealing the annulus with quick-set epoxy. The outlet of the foam generator is then connected to the inlet of the optical adhesive device using a 23-gauge connector.</li>
		<li>Connect the oil-filled syringe to the inlet of the device by inserting the needle tip into the PE/3 tubing.</li>
		<li>Begin flowing the oil into the outlet port of the OPTICAL ADHESIVE device at 2 mL/h while simultaneously flowing the displacing fluid into the inlet port at 0.8 mL/h such that the two fluids both flow out the drain port. The displacing fluid should not enter the porous media. Collect the effluent in a 20 mL glass vial.</li>
	</ol>
	</li>
	<li>Begin filming the AOI on the porous media device at a frame rate fast enough to capture the desired phenomena. A typical frame rate is 50 fps. Capture a still image of the 100% oil-saturated area.</li>
	<li>Swiftly and simultaneously cut the PE/3 tubing that is flowing in the oil using scissors while clamping the drain tube with a 5 cm binder clamp.</li>
	<li>Allow the displacing fluid to invade the device until either the oil displacement reaches steady-state or the camera runs out of memory.</li>
</ol>
5. Image and Data Analysis
<ol>
	<li>Use a free image analysis software such as Image J or use the image analysis toolbox in MATLAB to analyze the footage from the experiment.
	<ol>
		<li>Using the still image of the 100% oil-saturated channel, calculate the porosity in units of percent for the porous media AOI.</li>
	</ol>
	</li>
	<li>Calculate the pore volume using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/56592/56592eq1.jpg" /></li>
	<li>Use image analysis software to determine the oil saturation, as a fraction of the total flow space, in each frame of the video footage from the experiment. For two phase displacement experiments, the displacing phase saturation in each frame may be calculated as: 
	<img alt="Equation 2" src="/files/ftp_upload/56592/56592eq2.jpg" /></li>
	<li>Prepare a plot of oil saturation in percent vs. pore volumes of injected fluid 
	NOTE: Optional: For three phase systems such as those of foam displacement experiments, use the MATLAB image analysis toolbox to categorize each displacing phase by color using the characteristic RGB range for each phase. Prepare a plot showing the saturations of all three phases with injected pore volumes.</li>
</ol>

<ol>
	<li>Blaker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foam+for+Gas+Mobility+Control+in+the+Snorre+Field:+The+FAWAG+Project.">Foam for Gas Mobility Control in the Snorre Field: The FAWAG Project.</a> SPE Reserv Eval Eng. 5 (04), 317-323 (2002).</li><li>Mannhardt, K., Svorst&#248;l, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oil+saturation+on+foam+propagation+in+Snorre+reservoir+core.">Effect of oil saturation on foam propagation in Snorre reservoir core.</a> J Petrol Sci Eng. 23 (3-4), 189-200 (1999).</li><li>Falls, A. H., Lawson, J. B., Hirasaki, G. 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The authors have nothing to disclose.

This protocol for studying oil recovery processes in optical adhesive micromodels strikes a balance between the robustness of non-polymeric micromodels &#8211; such as glass or silicon &#8211; and the facile fabrication of PDMS microfluidic devices. Unlike micromodels made of glass or optical adhesive, PDMS devices lack resistance to light organic species. PDMS micromodels are also not ideal for many experiments because the surfaces of these devices have unstable wetting properties, and the polymer matrix is permeable to...

The purpose of this method is to visualize and analyze multi-phase, multi-component fluid interactions and complex pore-scale dynamics in porous media. Fluid flow and transport in porous media have been of interest for many years because these systems are applicable to several subsurface processes such as oil recovery, aquifer remediation, and hydraulic fracturing1,2,3,4,5. Using micromodels to mimic these complex pore-structures, unique insights are gained by visualizing pore-level dynamic events between the different fluid phases and the media6,7,8,9,10,11.
The fabrication of traditional silica-based micromodels is expensive, time consuming, and challenging, yet constructing micromodels from optical adhesive offers a relatively inexpensive, fast, and easy alternative12,13,14,15. Compared with other polymer-based micromodels, optical adhesive exhibits more stable surface wetting properties. For example, polydimethylsiloxane (PDMS) micromodel surfaces will quickly become hydrophobic during the course of a typical displacement experiment16. Furthermore, the Young&#39;s modulus of PDMS is 2.5 MPa whereas that of optical adhesive is 325 MPa13,17,18. Thus, optical adhesive is less prone to pressure induced deformation and channel failure. Importantly, cured optical adhesive is much more resistant to swelling by low molecular weight organic components, which allows experiments involving crude oil and light solvents to be conducted18. Overall, optical adhesive is a superior alternative to PDMS for displacement studies involving crude oil when silica-based micromodels are prohibitively complex or expensive and high temperature and pressure studies are not required.
The protocol described in this publication provides the step-by-step fabrication instructions for optical adhesive micromodels and reports the subtle tricks that ensure success in the manipulation of small quantities of fluids. The design and fabrication of optical adhesive based micromodels with soft lithography is first described. Then, the fluid displacement strategy is given for ultra-low flow rates that are commonly unattainable with mass flow controllers. Next, a representative experimental result is given as an example. This experiment reveals foam destabilization and propagation behavior in the presence of crude oil and heterogeneous porous media. Lastly, typical image processing and data analysis is reported.
The method provided here is appropriate for visualization applications involving multi-phase flow and interactions in confined microchannel spaces. Specifically, this method is optimized for characteristic micro-feature resolutions greater than 5 and less than 700 &#181;m. Typical flow rates are on the order of 0.1 to 1 mL/h. In studies of crude oil or light solvent displacement by aqueous or gaseous fluids on the order of these optimized parameters at ambient conditions, this protocol should be appropriate.

<table>
	<tbody>
		<tr>
			<td>3 mL Leur-Lok Syringe</td>
			<td>Fischer Scientific</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Glass Syringe</td>
			<td>Fischer Scientific</td>
			<td>1482698G</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask</td>
			<td>CAD/Art Services</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene-Glycol-Methyl-Ether-Acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>484431-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm Glass Petri Dish</td>
			<td>Carolina Biological Supply</td>
			<td>#721134</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Plastic Petri Dish</td>
			<td>Carolina Biological Supply</td>
			<td>#741246</td>
			<td></td>
		</tr>
		<tr>
			<td>Mask Aligner</td>
			<td>EV Group</td>
			<td>EVG 620</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm Biopsy Punch</td>
			<td>Miltex, Plainsboro, NJ</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Industrial Dispensing Tip</td>
			<td>CML Supply</td>
			<td>Gauge 23</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma System</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td>Plasma cleaner</td>
		</tr>
		<tr>
			<td>Polydimehtylsiloxane (PDMS)</td>
			<td>Dow Corning, Midland, MI</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Norland Optical Adhesive 81 (NOA81) or (OA)</td>
			<td>Norland Products Inc.</td>
			<td>8116</td>
			<td>Optical adhesive</td>
		</tr>
		<tr>
			<td>Quick-Set Epoxy</td>
			<td>Fisher Scientific</td>
			<td>4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>Globe Scientic Inc.</td>
			<td>1321</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2015 Photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2015</td>
			<td>Photo resist</td>
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			<td>Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>Fusion 400</td>
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			<td>Glass Capillary Tubing</td>
			<td>SGE Analytical Science</td>
			<td>1154710C</td>
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			<td>High-Speed Camera</td>
			<td>Vision Research</td>
			<td>V 4.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing</td>
			<td>Scientific Commodities Inc.</td>
			<td>#BB31695-PE/3</td>
			<td></td>
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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.

In this example experiment, aqueous foam is used to displace Middle East crude oil (with a viscosity of 5.4 cP and API gravity of 40&#176;) in a heterogeneous porous media with layered permeability contrast. A PDMS foam generator is connected to an optical adhesive micromodel which was previously completely saturated with crude oil. Figure 1a shows the CAD design of the photomask for the PDMS foam generator, the photoresist-patterned silicon wafer, and the co...

Watch this Scientific Journal Video about Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications at JoVE.com

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

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 ...

microfluidic-devices-for-characterizing-pore-scale-event-processes

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10.3K Views.  Rice University. 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.

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

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 ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Metal Corrosion and the Efficiency of Corrosion Inhibitors in Less Conductive Media

Material corrosion can be a limiting factor for different materials in many applications. Thus, it is necessary to better understand corrosion processes, prevent them and minimize the damages associated with them. One of the most important characteristics of corrosion processes is the corrosion rate. The measurement of corrosion rates is often very difficult or even impossible especially in less conductive, non-aqueous environments such as biofuels. Here, we present five different methods for the determination of corrosion rates and the efficiency of anti-corrosion protection in biofuels: (i) a static test, (ii) a dynamic test, (iii) a static test with a reflux cooler and electrochemical measurements (iv) in a two-electrode arrangement and (v) in a three-electrode arrangement. The static test is advantageous due to its low demands on material and instrumental equipment. The dynamic test allows for the testing of corrosion rates of metallic materials at more severe conditions. The static test with a reflux cooler allows for the testing in environments with higher viscosity (e.g., engine oils) at higher temperatures in the presence of oxidation or an inert atmosphere. The electrochemical measurements provide a more comprehensive view on corrosion processes. The presented cell geometries and arrangements (the two-electrode and three-electrode systems) make it possible to perform measurements in biofuel environments without base electrolytes that could have a negative impact on the results and load them with measurement errors. The presented methods make it possible to study the corrosion aggressiveness of an environment, the corrosion resistance of metallic materials, and the efficiency of corrosion inhibitors with representative and reproducible results. The results obtained using these methods can help to understand corrosion processes in more detail to minimize the damages caused by corrosion.

The testing of processes associated with material corrosion can often be difficult especially in non-aqueous environments. Here, we present different methods for short-term and long-term testing of corrosion behavior of non-aqueous environments such as biofuels, especially those containing bioethanol.

Material corrosion can be a limiting factor for different materials in many applications. Thus, it is necessary to better understand corrosion processes, ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...