Secondary flow as a mechanism for the formation of biofilm streamers

Abstract

In most environments, such as natural aquatic systems, bacteria are found predominantly in self-organized sessile communities known as biofilms. In the presence of a significant flow, mature multispecies biofilms often develop into long filamentous structures called streamers, which can greatly influence ecosystem processes by increasing transient storage and cycling of nutrients. However, the interplay between hydrodynamic stresses and streamer formation is still unclear. Here, we show that suspended thread-like biofilms steadily develop in zigzag microchannels with different radii of curvature. Numerical simulations of a low-Reynolds-number flow around these corners indicate the presence of a secondary vortical motion whose intensity is related to the bending angle of the turn. We demonstrate that the formation of streamers is directly proportional to the intensity of the secondary flow around the corners. In addition, we show that a model of an elastic filament in a two-dimensional corner flow is able to explain how the streamers can cross fluid streamlines and connect corners located at the opposite sides of the channel.

Figure 1

(a) Microfluidic experimental setup used to study biofilm formation under continuous flow and constant nutrient conditions. The channels are made in PDMS and sealed over a glass surface, which allows for a direct optical investigation (not to scale). (b) Confocal microscopy images taken in the middle-horizontal plane of the channel after 12 h of constant flow rate (from left to right) at 0.75 μl min⁻¹ for two different experiments. The bacteria are green fluorescent protein-labeled P. aeruginosa strain PA14. Scale bars: 100 μm. (c) Pictorial visualization of the suggested mechanism of streamer formation. The streamer is a 3D rendering from confocal image stacks, and the secondary flow contour plots were obtained from 3D numerical simulations of the flow.

Figure 2

Experimental design used to investigate the influence of the secondary vortical motion associated with different curvatures of the channel on the process of biofilm streamer formation. The overall length of the microfluidic channel is ∼88.5 mm, including 10 curved sections (such as the one highlighted in the box) of different internal angles spanned by the fluid, i.e., ${\alpha }_{fl}=210°,240°,270°,300°,330°$. The channel has a typical width (W) and height (H) of 200 μm and 85 μm, respectively.

Figure 3

Contour plots from numerical simulations of the velocity component u_z in a plane at 1/4 of the channel height from the bottom surface, for the angles ${\alpha }_{fl}=210°\left(a\right);270°\left(b\right);330°\left(c\right)$. White and dark gray contours indicate positive and negative values of u_z, respectively, and gray contours represent vanishing values of u_z. (d) Distribution of u_z (normalized with respect to the mean speed in the channel, U) along the x axis (normalized with respect to W) for different curvatures of the corner. The distances $\overline{y}$ and $\overline{z}$ from the side wall and the bottom of the channel, respectively, were chosen such that $u_z/U$ has the maximum value (shown in the inset as a function of α_fl). The direction of the flow is from left to right.

Figure 4

(a) Biofilm streamers that developed after 18 h of continuous flow, from left to right, at 0.5 μl min⁻¹ (OD₆₀₀$=0.25$). The confocal image sequences on the left and right sides were obtained from the bottom and mid-horizontal planes of the channel, respectively, for all of the curved sections shown in Fig. 2. Scale bars: 200 μm. (b) Average diameter of the streamers after ∼14 h of continuous flow versus α_fl for different flow rates, i.e., 0.5 μl min⁻¹$\left(\mathrm{▾}\right)$, 1 μl min⁻¹$\left(\mathrm{■}\right)$, and 1.5 μl min⁻¹$\left(\mathrm{▴}\right)$.

Figure 5

(a) Time-lapse confocal images of the formation of streamers for three bending angles: ${\alpha }_{fl}=270°,300°,330°$(from top to bottom). Each image was acquired in the middle horizontal plane of the channel at a flow rate of 1 μl min⁻¹. The initial concentration of bacteria was equal to OD₆₀₀$=0.1$. Scale bars: 200 μm. (b) Onset time (t₀) for the formation of streamers as a function of α_fl. The data shown represent different sets of experiments performed at flow rates of 0.5 μl min⁻¹ (▾), 1 μl min⁻¹ (■), and 1.5 μl min⁻¹ (▴), with the same initial concentration of bacteria in solution (OD₆₀₀$=0.1$). The inset shows the same data plotted as $t_0{u}_z^{max}/U$ versus the normalized maximum velocity perpendicular to the main flow direction, $u_z^{max}/U$, where $u_z^{max}$ is the velocity component obtained with the numerical simulations.

Figure 6

Log-linear plots of the average streamer diameter $\langle d\rangle$ as a function of time for different bending angles: ${\alpha }_{fl}=330°(●)$, $300°(○)$, and $270°(\oplus )$. The flow rates were equal to 1 μl min⁻¹ and 0.5 μl min⁻¹ (inset), given the same bacterial concentration (OD₆₀₀$=0.1$).

Figure 7

Viscoelastic properties of the streamers. Solid lines represent 4th-order polynomial interpolations of the shape of the filaments. (a) Image superimposition of a streamer at different flow rates (without flow, 0.25 μl min⁻¹, and 0.5 μl min⁻¹). (b) Image superimposition of the streamer without flow and with flow rate at 1 μl min⁻¹ after 30, 150, and 300 s, respectively, from the condition at rest. (c) Main graph: Stress-strain relationship for different streamers, with average diameters of ∼9.7 μm $\left(\mathrm{▾}\right)$, 21.3 μm $\left(\mathrm{■}\right)$, and 5.4 μm $(●)$. The filaments were all located in the middle horizontal plane of the channel while two consecutive corners were connected. Horizontal and vertical error bars represent variation in the strain and stress for the same flow rate over different tests. The effective elastic modulus, obtained from a linear fit of the data (dashed lines), varies in a range between 70 and 140 Pa. Inset: Streamer deformation as a function of time at viscous stresses of 5.7, 17, and 41 Pa applied for ∼600, 1500, and 600 s, respectively (interspersed with 30 s without flow). Dashed lines represent linear fits of the data during the flow conditions.

Figure 8

(a–c) Numerical results for a flexible filament in a viscous 2D flow around a corner. Steady-state positions for different lengths of the filament are shown. The values of elasticity, thickness of the filament, and Reynolds number used in the numerical model were 100 Pa, 1 μm, and 0.001, respectively. (d) Experimental biofilm streamer after 18 h of continuous flow at 0.5 μl min⁻¹.