Name: a microfluidic platform to study bioclogging in porous media
Uploaded: 2022-10-13T15:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform to Study Bioclogging in Porous Media at JoVE.com

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>
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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>
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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 ...

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Environment

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