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 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.
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' 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) 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 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.
1. Silicon wafer preparation
2. Fabrication of the microfluidic device
NOTE: The fabrication procedure described here is for a microfluidic device with one microfluidic channel. However, the same method can be applied to fabricate a microfluidic device with multiple microfluidic channels in parallel.
3. Preparation of the bacterial suspension
4. Biofilm growth experiment
5. Image analysis
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 biofilm colonized almost the entire porous medium.
The surface coverage of the biofilm in time grown at a flow rate of Q = 1 mL/h, which corresponds to a mean initial fluid flow velocity of 0.96 mm/s, was quantified for three different pore sizes (75 µm, 150 µm, and 300 µm) (Figure 3, black lines). It was found that the surface coverage, which was used as a proxy for the bioclogging degree, occurred 10% faster at the smallest pore size of 75 µm than at the biggest pore size (300 µm) when comparing the surface coverage at t = 20 h. Then, the surface coverage was correlated to the pressure buildup caused by the biofilm (Figure 3, blue lines). 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 microfluidic channels, indicating that smaller-sized porous media will develop higher pressure buildup when subjected to bioclogging.
Figure 1: Microfluidic channel design and experimental setup. (A) Photomask of the microfluidic channels with different pore sizes (75 µm, 150 µm, and 300 µm) used as porous media analogs and a zoomed-in view of the pillars' arrangement (bottom row). The circles show the location of the pillars (impermeable obstacles), representing the porous media's solid phase. (B) Schematic of the experimental setup showing the syringe, the pressure sensor, the microfluidic device (with a single microfluidic channel), and the digital camera setup with the objective (i.e., the microscope). Please click here to view a larger version of this figure.
Figure 2: Visualization and quantification of biofilm development in the porous medium. (A) Representative image sequence of the biofilm development at the imposed flow rate of Q = 1 mL/h (corresponds to a mean initial fluid flow velocity of 0.96 mm/s) and a pore size of d = 300 µm shown for the experimental time points t = 5 h, t = 10 h, t = 15 h, and t = 20 h. The bright-field images were stitched, and the background was removed. (B) The binarization of these images and quantification of the area occupied by biofilm (dark pixels) led to the quantification of surface coverage in Figure 3. Please click here to view a larger version of this figure.
Figure 3: Temporal evolution of biofilm coverage and impact on pressure. Biofilm coverage with simultaneous pressure reading for the three pore sizes (300 µm, 150 µm, and 75 µm) in the same experimental conditions as Figure 2. The pressure difference caused by the biofilm in the porous medium microfluidic channel, Δp, (blue lines) shown on the right y-axis, increases with an increased surface coverage of the biofilm (black lines). The green markers correspond to the data points of the images shown in Figure 2. Please click here to view a larger version of this figure.
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 media. In the present microfluidic platform, three porous media geometries were created with regularly arranged cylindrical pillars (pore sizes: 75 µm, 150 µm, and 300 µm), where the fluid flow rate could be chosen per experiment. The presented platform can be easily adapted to study bioclogging with a fixed pressure head rather than an imposed fluid flow rate. In this case, the flow control device should be a pressure controller with a culture medium reservoir instead of a syringe pump. The resulting changes in flow rate due to bioclogging could be monitored by measuring the outflow over time using a flow rate sensor.
Several critical points must be considered to run a successful microfluidic experiment with biofilm growth. To avoid air bubble formation in the microfluidic channel during the experiment, the microfluidic channel and the culture medium were degassed (step 4.3). Next, filling of the microfluidic channel with the degassed culture medium must be conducted rapidly but carefully to obtain a fully saturated channel without any air bubbles. In case air bubbles are trapped in the porous medium, flushing the microfluidic channel at a higher flow rate can remove the bubbles after a short time. The second crucial step is to ensure a constant temperature environment to reproduce biofilm growth consistently. The growth of microorganisms varies with temperature25, which might lead to non-reproducible results when not keeping the temperature stable during the experiment (in this case, 24 h). For the present platform, a box incubator was used around the microscope, though a smaller temperature-stable casing for the microfluidic device would likely be sufficient too. Finally, during the image acquisition, the positions of the individual images should be chosen with an overlap of at least 15% to obtain enough overlap for the stitching algorithm24.
The present microfluidic platform is limited to two-dimensional observation, whereas porous media applications like soil or membranes have a three-dimensional structure. However, advantages of the quasi-2D microfluidic platform compared to 3D porous media platforms to study bioclogging are the full optical access and the high time resolution, as 3D platforms usually perform endpoint imaging26,27. In addition, it is expected that the bioclogging process (i.e., the time evolution of surface coverage) persists in 3D systems26,27, as it also occurs for the cluster size distribution of an immiscible phase within porous media28, which presents the same scaling in 2D and 3D systems.
This method allows measuring the pressure response to biofilm growth in porous media while studying its spatio-temporal development at high temporal and spatial resolution and different pore sizes. The data sets obtained from such measurements bring insight into the correlation of pore-scale biofilm development with pressure responses of the biofilm-porous medium system, and can provide a benchmark for the numerical modeling of biofilms. These modeling efforts are especially relevant to extending the range of conditions (e.g., pore sizes, flow velocities, and biofilm properties for other species or multispecies biofilms) that exceed experimental capacities. The latter is highly relevant to understanding the mechanisms of bioclogging in the vicinity of wells, bioremediation applications, and biomineralization29,30,31. Overall, this method could easily be adapted to study biomineralization or track the biotransformation of contaminants by biofilms in porous media.
The authors have nothing to disclose.
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.
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 |