A Microfluidic Platform to Study Bioclogging in Porous Media

Summary

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.

Abstract

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.

Introduction

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 DNA^5,6, and strongly depends on the microorganisms, the availability of nutrients, as well as the environmental conditions⁷. Yet, the fun....

Protocol

1. Silicon wafer preparation

1. 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).
2. Fabricate the master mold by soft lithography (under clean-room conditions) following the steps below.
   1. Bake the silicon wafer at 200 °C for 2 h.
   2. Place the wafer at the center of a spin-coater and pour SU8 3050 photoresist (see Table of Materials) onto the wafer. Spin coat at 1,700 rpm for 40 s with a 10 s/100 rpm ramp time.

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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