Early biofilm and streamer formation is mediated by wall shear stress and surface wettability: A multifactorial microfluidic study

Abstract

Biofilms are intricate communities of microorganisms encapsulated within a self-produced matrix of extra-polymeric substances (EPS), creating complex three-dimensional structures allowing for liquid and nutrient transport through them. These aggregations offer constituent microorganisms enhanced protection from environmental stimuli-like fluid flow-and are also associated with higher resistance to antimicrobial compounds, providing a persistent cause of concern in numerous sectors like the marine (biofouling and aquaculture), medical (infections and antimicrobial resistance), dentistry (plaque on teeth), food safety, as well as causing energy loss and corrosion. Recent studies have demonstrated that biofilms interact with microplastics, often influencing their pathway to higher trophic levels. Previous research has shown that initial bacterial attachment is affected by surface properties. Using a microfluidic flow cell, we have investigated the relationship between both wall shear stress (τ_w ) and surface properties (surface wettability) upon biofilm formation of two species (Cobetia marina and Pseudomonas aeruginosa). We investigated biofilm development on low-density polyethylene (LDPE) membranes, Permanox® slides, and glass slides, using nucleic acid staining and end-point confocal laser scanning microscopy. The results show that flow conditions affect biomass, maximum thickness, and surface area of biofilms, with higher τ_w (5.6 Pa) resulting in thinner biofilms than lower τ_w (0.2 Pa). In addition, we observed differences in biofilm development across the surfaces tested, with LDPE typically demonstrating more overall biofilm in comparison to Permanox® and glass. Moreover, we demonstrate the formation of biofilm streamers under laminar flow conditions within straight micro-channels.

Keywords: Cobetia marina; Pseudomonas aeruginosa; biofilm; biofilm formation; biofilm streamers; biofouling; microfluidics; surface wettability; wall shear stress.

Figure 1

Schematic showing the design elements of the microfluidic flow cell, where (a) the span of the microfluidic channels, (b) top‐down view of the channels, (c) cross‐sectional view of the channel, showing the step‐like progression of the chambers, (d) the mesh size and total elements in the flow cell design, features critical to the CFD calculations, and (e) the wall shear stress over the bottom surface of the chambers as determined from the numerical simulations. CFD, computational fluid dynamic

Figure 2

Schematic illustrating the experimental set‐up, showing the constituent parts including an image of the flow cell. Arrows illustrate the direction of flow. Dotted lines indicate the option of adding a CCD camera for real‐time measurements.

Figure 3

Influence of shear stress on Cobetia marina on the left and Pseudomonas aeruginosa on the right‐hand side when exposed to different surfaces. Results collected from each test surface are compiled above including the R² value for the linear fit, in the order of LDPE, Permanox® and Glass, respectively. Error bars ± SE. Note: the associated statistical analysis can be found in Tables A2, A3, A4, A5. SE, standard error

Figure 4

Confocal laser scanning microscopy (CLSM) images of Cobetia marina and Pseudomonas aeruginosa biofilm on low‐density polyethylene (LDPE), Permanox®, and glass showing both the XY and XZ planes. In all images, the flow was oriented from left to right, with scale bars of 50 µm. These images demonstrate that overall biomass and biofilm thickness decrease as the wall shear stress levels increase.

Figure 5

Biofilm streamers recorded under the higher wall shear stress level of 5.6 Pa. Endpoint images of (a) Cobetia marina on Permanox®, (b) Cobetia marina on glass, and (c) Pseudomonas aeruginosa on the glass; streamers indicated by arrows. Images were captured using a 63X objective lens; scale bars are 50 µm. Flow is oriented from left to right in all images. Note: These images were taken under static conditions.

Figure A1

Vertical profiles for Cobetia marina biofilm on LDPE showing the percentage coverage in each slice of the Z‐stacks. LDPE, low‐density polyethylene

Figure A2

Vertical profiles for Cobetia marina biofilm on Permanox® showing the percentage coverage in each slice of the Z‐stacks.

Figure A3

Vertical profiles for Cobetia marina biofilm on Glass showing the percentage coverage in each slice of the Z‐stacks and distance from the surface.

Figure A4

Vertical profiles for Pseudomonas aeruginosa biofilm on LDPE showing the percentage coverage in each slice of the Z‐stacks and the distance from the surface. LDPE, low‐density polyethylene

Figure A5

Vertical profiles for Pseudomonas aeruginosa biofilm on Permanox® showing the percentage coverage in each slice of the Z‐stacks and the distance from the surface.

Figure A6

Vertical profiles for Pseudomonas aeruginosa biofilm on Glass showing the percentage coverage in each slice of the Z‐stacks and the distance from the surface.

Figure A7

Streamer images recorded on LDPE using confocal laser scanning microscopy with a 63x objective lens, scale bars are 50 µm. Flow is orientated from left to right. (a) shows Cobetia marina (b) shows Pseudomonas aeruginosa. LDPE, low‐density polyethylene