The authors acknowledge support from SNSF PRIMA grant 179834 (to E.S.), discretionary funding from ETH (to R.S.), ETH Zurich Research Grant (to R.S. and J.J.M.), and discretionary funding from Eawag (to J.J.M.). The authors would like to thank Roberto Pioli for illustrating the experimental setup in Figure 1B and Ela Burmeister for the silicon wafer preparation.

1. Silicon wafer preparation
<ol>
	<li>Design the geometries of the microfluidic channel in computer-aided design (CAD; see Table of Materials) software and print it onto a transparent film to create the photomask (Figure 1A).</li>
	<li>Fabricate the master mold by soft lithography (under clean-room conditions) following the steps below.
	<ol>
		<li>Bake the silicon wafer at 200 &#176;C for 2 h.</li>
		<li>Place the wafer at the center of a spin-coater and...

<ol>
	<li>Flemming, H. C., Wuertz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+archaea+on+Earth+and+their+abundance+in+biofilms.">Bacteria and archaea on Earth and their abundance in biofilms.</a> Nature Reviews Microbiology. 17 (4), 247-260 (2019).</li><li>Cunningham, A. B., Sharp, R. R., Hiebert, R., James, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsurface+biofilm+barriers+for+the+containment+and+remediation+of+contaminated+groundwater.">Subsurface biofilm barriers for the containment and remediation of contaminated groundwater.</a> Bioremediation Journal. 7 (3-4), 151-164 (2003).</li><li>Pronk, W., 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=Gravity-driven+membrane+filtration+for+water+and+wastewater+treatment:+A+review.">Gravity-driven membrane filtration for water and wastewater treatment: A review.</a> Water Research. 149, 553-565 (2019).</li><li>Caldara, M., Belgiovine, C., Secchi, E., Rusconi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental,+microbiological,+and+immunological+features+of+bacterial+biofilms+associated+with+implanted+medical+devices.">Environmental, microbiological, and immunological features of bacterial biofilms associated with implanted medical devices.</a> Clinical Microbiology and Infection. 35 (2), 00221 (2022).</li><li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nature Reviews Microbiology. 8 (9), 623-633 (2010).</li><li>Davey, M. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms:+from+ecology+to+molecular+genetics.">Microbial biofilms: from ecology to molecular genetics.</a> Microbiology and Molecular Biology Reviews. 64 (4), 847-867 (2000).</li><li>Stoodley, P., Dodds, I., Boyle, J. D., Lappin-Scott, H. 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L., Scott Fogler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore-scale+investigation+of+biomass+plug+development+and+propagation+in+porous+media.">Pore-scale investigation of biomass plug development and propagation in porous media.</a> Biotechnology and Bioengineering. 77 (5), 577-588 (2002).</li><li>Hassanpourfard, M., Ghosh, R., Thundat, T., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bacterial+streamers+induced+clogging+in+microfluidic+devices.">Dynamics of bacterial streamers induced clogging in microfluidic devices.</a> Lab on a Chip. 16 (21), 4091-4096 (2016).</li><li>Aufrecht, J. A., 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=Pore-scale+hydrodynamics+influence+the+spatial+evolution+of+bacterial+biofilms+in+a+microfluidic+porous+network.">Pore-scale hydrodynamics influence the spatial evolution of bacterial biofilms in a microfluidic porous network.</a> PLoS ONE. 14 (6), 0218316 (2019).</li><li>Karimifard, S., Li, X., Elowsky, C., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+impact+of+evolving+biofilms+on+flow+in+porous+media+inside+a+microfluidic+channel.">Modeling the impact of evolving biofilms on flow in porous media inside a microfluidic channel.</a> Water Research. 188, 116536 (2021).</li><li>Yawata, Y., Nguyen, J., Stocker, R., Rusconi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+studies+of+biofilm+formation+in+dynamic+environments.">Microfluidic studies of biofilm formation in dynamic environments.</a> Journal of Bacteriology. 198 (19), 2589-2595 (2016).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Ratkowsky, D. A., Olley, J., McMeekin, T. 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Microfluidic porous media analogs coupled with pressure sensors provide a suitable tool to study biofilm development in porous media. The versatility in the design of the microfluidic porous medium, specifically the arrangement of the pillars, including diameter, irregular shapes, and pore size, allows the investigation of many geometries. These geometries range from single pores to highly complex, irregularly arranged obstacles mimicking different natural (e.g., soils) and industrial (e.g., membranes and filters) porous...

Biofilms - bacterial colonies embedded in a self-secreted matrix of extra-polymeric substances (EPS) - are ubiquitous in natural porous media, such as soils and aquifers1, and technical and medical applications, like bioremediation2, water filtration3 and medical devices4. The biofilm matrix is comprised of polysaccharides, protein fibers, and extracellular DNA5,6, and strongly depends on the microorganisms, the availability of nutrients, as well as the environmental conditions7. Yet, the functions of the matrix are universal; it forms the scaffold of the biofilm structure, protects the microbial community from mechanical and chemical stresses, and is largely responsible for the biofilms&#39; rheological properties5.
In porous media, the growth of biofilms can clog pores, causing the so-called bioclogging. Biofilm development is controlled by the fluid flow and pore size, defined as the distance separating two pillars, of the porous medium8,9,10. Both the pore size and the fluid flow control the nutrient transport and local shear forces. In turn, the growing biofilm clogs pores, affecting the velocity distribution of the fluid11,12,13, the mass transport, and the hydraulic conductivity of the porous medium14,15. The changes in hydraulic conductivity are reflected through increased pressure in confined systems16,17,18,19. Current microfluidic studies in biofilm development and bioclogging focus on studying the impact of flow velocities in homogeneous geometries16,20 (i.e., with a singular pore size)&#160;or heterogeneous porous media12,21,22. However, to disentangle the effects of flow rates and pore size on biofilm development and the resulting pressure changes in the bioclogged porous medium, a highly controllable and versatile experimental platform allowing the study of different porous media geometries and environmental conditions in parallel is required.
The present study introduces a microfluidic platform that combines pressure measurements with simultaneous imaging of the evolving biofilm within the porous medium. Because of its gas-permeability, bio-compatibility, and flexibility in the channel geometry design, a microfluidic device made of polydimethylsiloxane (PDMS) is a suitable tool for studying biofilm development in porous media. Microfluidics allow&#160;the control of physical and chemical conditions (e.g., fluid flow and nutrient concentration) with high precision to mimic the environment of microbial habitats23. Further, microfluidic devices can easily be imaged with micrometric resolution using an optical microscope and coupled with online measurements (e.g., the local pressure).
In this work, the experiments focus on studying the impact of pore size in a homogeneous porous medium analog under controlled imposed flow conditions. The flow of a culture medium is imposed using a syringe pump, and the pressure difference through the microfluidic channel is measured simultaneously with pressure sensors. Biofilm development is initiated by seeding a planktonic culture of Bacillus subtilis in the microfluidic channel. Regular imaging of the evolving biofilm and image analysis allows one to obtain pore scale resolved information on the surface coverage under various experimental conditions. The correlated information of pressure change and the extent of bioclogging provides crucial input for permeability estimations of bioclogged porous media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc 25 mm Syringe Filter, 1.2 &#181;m Versapor Membrane</td>
			<td>Pall Corporation</td>
			<td>PN4190</td>
			<td>1.2 &#181;m filters</td>
		</tr>
		<tr>
			<td>BD 10 mL Syringe (Luer-Lock)</td>
			<td>BD</td>
			<td>300912</td>
			<td>used to fill the channel with deionised water</td>
		</tr>
		<tr>
			<td>Box Incubator</td>
			<td>Life Imaging Services</td>
			<td></td>
			<td>used to have a stable temperature during the biofilm growth experiment</td>
		</tr>
		<tr>
			<td>Cell density meter CO8000</td>
			<td>WPA biowave</td>
			<td></td>
			<td>OD meter</td>
		</tr>
		<tr>
			<td>Centrifuge vial</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>CETONI Base 120</td>
			<td>CETONI GmbH</td>
			<td></td>
			<td>syringe pump</td>
		</tr>
		<tr>
			<td>CorelCAD</td>
			<td>CorelDRAW</td>
			<td></td>
			<td>software used to design the microfluidic channel geometries</td>
		</tr>
		<tr>
			<td>Culture tubes (14 mL, sterile)</td>
			<td>greiner bio-one</td>
			<td></td>
			<td>Culture tubes</td>
		</tr>
		<tr>
			<td>Drying oven, VENTI-Line</td>
			<td>VWR</td>
			<td></td>
			<td>Oven to cure the PDMS</td>
		</tr>
		<tr>
			<td>Handy</td>
			<td>Migros</td>
			<td></td>
			<td>Detergent solution</td>
		</tr>
		<tr>
			<td>Hot plate with temperature control</td>
			<td>VRW</td>
			<td></td>
			<td>to cure the PDMS-glass bonding after plasma treatment</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>FIJI&#160;</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Innova 42 Inc Shaker (New Brunswick)</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Incubator</td>
		</tr>
		<tr>
			<td>Isopropanol (&#62; 99.8%)</td>
			<td>Sigma Aldrich</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex transfer tubing</td>
			<td>Masterflex</td>
			<td>HV-06419-05</td>
			<td>0.020&#39;&#39; ID, 0.06&#39;&#39; OD</td>
		</tr>
		<tr>
			<td>Micro Slides, Plain, 75 x 60 mm</td>
			<td>Corning</td>
			<td>2947-75X50</td>
			<td>Glass slides</td>
		</tr>
		<tr>
			<td>Microfluidic pressure sensor (1 bar)</td>
			<td>Elveflow</td>
			<td></td>
			<td>Pressure sensors</td>
		</tr>
		<tr>
			<td>Miltex Biopsy puncher, diameter 1.5 mm</td>
			<td>Integra</td>
			<td></td>
			<td>Puncher to make the inlet and outlet holes of the microfluidic channel</td>
		</tr>
		<tr>
			<td>mrDev600 developer</td>
			<td>Microresist</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Nutrient broth n&#176;3</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix Syringe with Luer-Lock</td>
			<td>B.Braun</td>
			<td></td>
			<td>syringes of different volume</td>
		</tr>
		<tr>
			<td>Plasma chamber Zepto</td>
			<td>Diener Electronic</td>
			<td>ZEPTO-1&#160;</td>
			<td>used to plasma bond the PDMS and the glass slide</td>
		</tr>
		<tr>
			<td>Precision wipes (Kimtech Science)</td>
			<td>Kimberly Clark</td>
			<td>KCP-7552</td>
			<td>to dry the glass slide</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>VWR-CH</td>
			<td>611-2605</td>
			<td>used to weigh the elastomer to crosslinking agent ratio</td>
		</tr>
		<tr>
			<td>Silicon wafer (10 cm)</td>
			<td>Silicon Materials Inc.&#160;</td>
			<td></td>
			<td>N//Phos &#60;100&#62; 1-10 &#937; cm</td>
		</tr>
		<tr>
			<td>Spincoater, Spin module SM150</td>
			<td>Sawatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU8 3050 Photoresist</td>
			<td>Kayakuam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S&#252;ss MA6 Mask aligner</td>
			<td>SUSS MicroTec Group</td>
			<td></td>
			<td>used to align the chrome-glass mask</td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td>silicone elastomer kit; curing agent</td>
		</tr>
		<tr>
			<td>Techni Etch Cr01</td>
			<td>Technic</td>
			<td></td>
			<td>Technic</td>
		</tr>
		<tr>
			<td>Tissue culture dish 150</td>
			<td>TPP</td>
			<td>93150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane</td>
			<td>Sigma Aldrich</td>
			<td>Sigma Aldrich</td>
			<td>used to silanize the silicane wafer</td>
		</tr>
		<tr>
			<td>Veeco Dektak 6 M</td>
			<td>Veeco</td>
			<td></td>
			<td>Profilometer</td>
		</tr>
	</tbody>
</table>

a microfluidic platform to study bioclogging in porous media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

For the present study, a microfluidic device with three parallel microfluidic channels with different pore sizes was used (Figure 1) to study biofilm formation in porous media systematically. The biofilm formation process was visualized using bright-field microscopy. The bacterial cells and the biofilm appeared in the images as darker pixels (Figure 2). In addition, a gradual clogging process was observed; during a 24 h experiment, the initially randomly growing...

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A Microfluidic Platform to Study Bioclogging in Porous Media

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

This method allows the systematic study of the influence of pore size and flow rate on biofilm development in natural as well as artificial porous media, thereby fundamental mechanism of porous media bioclogging can be unraveled. The main advantages of the techniques are the highest spatial and temporal resolution of the alteration, and adaptability of the platform to a range of experimental conditions and requirements. Begin by turning on the box incubator of the microscope three hours before the experiment to ensure a stable temperature of 25 degrees Celsius.Connect the inlet and outlet tubing to the microfluidic device. Secure the connection between the tubing and the syringe by inserting a needle having an outer diameter of 0.6 millimeters into the inlet tubing. Place the microfluidic device, 30 milliliters of deionized water, and 30 milliliters of culture medium in a vacuum desiccator and degas them for at least one hour.Once done, slowly pull the culture medium and the deionized water into two separate 30-milliliter syringes. Then, mount the microfluidic device on the microscope and place the outlet tubing in a waste container. Connect the syringe filled with deionized water to the microfluidic channel through the microfluidic tubing and slowly inject the water until it exits from the pressure sensor outlet.Fill the pressure sensor with water and flush all the bubbles from the tubing connecting the microfluidic channel and the pressure sensor. Close the outlet of the pressure sensor with the screws dedicated to the pressure sensor. Fill the rest of the microfluidic channel with deionized water.Next, place a 1.2-micrometer filter on the culture media syringe. Remove the water syringe and carefully connect the syringe to the inlet microfluidic tubing. After mounting the syringe on the syringe pump, flush the channel with the culture medium at a flow rate of two milliliters per hour for one hour.Thereafter, set the syringe pump to the desired flow rate during the experiment and set the pressure reading of the pressure sensors to zero. Next, pipette one milliliter of the Bacillus subtilis NCIB 3610 culture of an optical density of 0.1 at a 600 nanometer wavelength in a 1.5-milliliter centrifuge vial. To load the bacterial culture into the microfluidic channel, place the outlet tube into the centrifuge vial and wait for five minutes to remove any potential air bubbles from the tube's outlet.Once done, withdraw 150 microliters of bacterial solution at a flow rate of one milliliter per hour until the microfluidic channel is filled with the bacterial culture. Then, carefully remove the culture media syringe filter and place the outlet into the waste container. Leave the bacterial cells at zero-flow conditions in the microfluidic channel for three hours to allow their surface attachment in the porous medium.To start the experiment, start the flow by setting the syringe pump to the desired flow rate and start the pressure reading at one hertz before acquiring the images of the growing biofilms at the desired time interval, optical configuration, and magnification. The biofilm formation process was visualized using bright-field microscopy, where the bacterial cells and the biofilm appeared in the images as darker pixels. During a 24-hours experiment, the initially randomly-growing biofilm colonized almost the entire porous medium.The surface coverage of the biofilm occurred 10%faster at the smallest pore size than at the biggest pore size at 20 hours. Correlation of the biofilm surface coverage with the pressure buildup showed that the clogging in the smaller pore size microfluidic channel led to a higher pressure difference between the inlet and the outlet than in the larger pore size. The most important aspects to remember are to run the experiment in a temperature stable environment and to degas the microfluidic device and the solutions well in order to avoid bubble formation.This method allows systematic studies to unravel fundamental mechanisms of biofilm growth in both industrial and natural porous media with regard to the influence of flow rate and pore size.

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Research

JoVE Journal

Environment

1.6K vistas.  ETH Zurich. Este método permite el estudio sistemático de la influencia del tamaño de poro y la velocidad de flujo en el desarrollo de biopelículas en medios porosos naturales y artificiales, por lo que se puede desentrañar el mecanismo fundamental de la bioobstrucción de medios porosos. Las principales ventajas de las técnicas son la mayor resolución espacial y temporal de la alteración, y la adaptabilidad de la plataforma a una gama de condiciones y requisitos experimentales. Comience encendie...