Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing

Abstract

Bacterial communities attached to surfaces under fluid flow represent a widespread lifestyle of the microbial world. Through shear stress generation and molecular transport regulation, hydrodynamics conveys effects that are very different by nature but strongly coupled. To decipher the influence of these levers on bacterial biofilms immersed in moving fluids, we quantitatively and simultaneously investigated physicochemical and biological properties of the biofilm. We designed a millifluidic setup allowing to control hydrodynamic conditions and to monitor biofilm development in real time using microscope imaging. We also conducted a transcriptomic analysis to detect a potential physiological response to hydrodynamics. We discovered that a threshold value of shear stress determined biofilm settlement, with sub-piconewton forces sufficient to prevent biofilm initiation. As a consequence, distinct hydrodynamic conditions, which set spatial distribution of shear stress, promoted distinct colonization patterns with consequences on the growth mode. However, no direct impact of mechanical forces on biofilm growth rate was observed. Consistently, no mechanosensing gene emerged from our differential transcriptomic analysis comparing distinct hydrodynamic conditions. Instead, we found that hydrodynamic molecular transport crucially impacts biofilm growth by controlling oxygen availability. Our results shed light on biofilm response to hydrodynamics and open new avenues to achieve informed design of fluidic setups for investigating, engineering or fighting adherent communities.

Fig 1. Shear stress significantly varies across the width of a millifluidic channel.

(A) Channel blueprint. (B) Bottom shear stress values, obtained by differentiation of the calculated velocity fields, are given for the 5 PDMS channels having a 30 mm length, 1 mm width (y axis) and 250 μm (red); 350 μm (orange); 500 μm (green); 750 μm (light blue); 1 mm (dark blue) height, under a flow rate of 1 ml/h.

Fig 2. Quantitative monitoring of biofilm growth reveals shear stress impact on biofilm development.

(A) Surface pattern of biofilm growing in the uniform growth mode (upper panel) and in the advancing-front growth mode (lower panel), yellow arrows indicate biofilm expansion directions; bright field picture taken using a 20x objective after 12 hours of continuous growth in M63B1 glucose medium in a 1 mm-height channel (uniform mode, upper panel) and a 250 μm-height channel (advancing-front mode, lower panel). The right edge of the picture coincides with the right edge of the channel, and a 330x440 μm² field is imaged. (B) Images are divided in 10 adjacent ROIs of 40 μm width, numbered from 1 (darker shade) to 10 (lighter shade); ROI1 is the channel edge ROI and ROI10 is a central ROI, located at 400 μm from the channel edge. (C) Bottom shear stress ranges overlapping between the 5 channels of the setup with channel heights on the left and aspect ratios on the right. Same channel color code as in Fig 1. (D) Series of growth curves derived from time-lapse image analysis. The microscopic absorbance, A_μ = ln(I₀/I), reporting the local biomass, is plotted as a function of time under continuous nutrient flow. Data points are represented in color for 10 ROIs per channel (one channel per graph) using the same color code as in Fig 1, and shades from dark to light coding for ROI1 to ROI10. Plot of a representative experiment of at least three (see data statistical dispersion in S1 Table). (E) Biofilm initiation is subjected to a 10 mPa shear stress threshold. Colonized ROIs (closed symbols) and non-colonized ones (open symbols), as observed in the 5 channels (same color code as in Fig 1) during the initial phase of biofilm development, are ordered on a shear stress range, evidencing the 10 mPa transition (arrow) between a shear stress which permits initial adhesion and a shear stress which does not.

Fig 3. Exponential adjustment and derivation of growth parameters.

A typical experimental curve showing microscopic absorbance (blue dots and left y axis) is represented together with the curve corrected by the microscopic absorbance-concentration relation in S1 Fig (red dots and right y axis) and the exponential adjustment (black line) giving the growth rate μ. The lag time δ is defined as the time where the corrected absorbance hits 0.02 (dashed line).

Fig 4. Below 10 mPa, shear stress does not affect biofilm growth rate, but impacts apparent lag time.

(A) Biofilm growth rate, μ (as derived from the exponential adjustment, see Fig 3) as a function of bottom shear stress. (B) The apparent lag time, δ, increases as a function of shear stress; experimental data (solid colored dots) can be adjusted using an exponential law (grey line). Data were collected from biofilm growing in the low shear stress regime, i.e. at σ <10 mPa. Data are represented using the same channel color code as in Fig 1. Means and SDs over two positions are represented. Plot of a representative experiment of at least three (see data statistical dispersion in S1 Table).

Fig 5. Local growth rate and lag time in the advancing-front growth mode characterize the biofilm spatial spreading.

Biofilm growth rate, μ (A) as derived from the exponential adjustment, (see Fig 3) and apparent lag time δ (B), as a function of the lateral location in the channels in the high bottom shear stress regime (> 10 mPa)—the 250 μm-height one (red dots) and the 350 μm-height one (orange dots). The slope of the lag time linear adjustment (grey line) provides the front propagation velocity. Means and SDs over two positions are represented. Plot of a representative experiment of at least three (see data statistical dispersion in S1 Table).

Fig 6. The velocity field evolves as the biofilm grows in the channel.

(A) Effective height of a 1 mm-total-height channel in the presence of growing biofilm. Main panel: Adjusted effective height h versus time t of biofilm growth. Error bars represent 95% confidence intervals on h, obtained from the adjustment. Insets: examples of adjustment of the velocity field. Dots: experimentally-measured velocities v at various positions (y, z) across the channel. Data was collected for all z within a narrow band of y (width Δy = 75 μm << w) located at the middle of the channel. Solid red curve: adjustment to the theoretical expression of the velocity field of a viscous fluid in a rectangular channel (Eq 1), plotted with y in the middle of the Δy-wide band. Dashed red curves: idem for y at each edge of the Δy-wide band. In addition to the effective height h, the volumetric flow rate Q was adjusted (through—η dp/dx), as in the absence of biofilm (S3 Fig), yielding Q = 0.85 mL/h, consistent with the adjusted value Q = 0.83 mL/h obtained in the biofilm-free case (S3 Fig). (B) Apparent size reduction in a 250 μm-height channel colonized by a biofilm after 15h growth. Dots: experimentally-measured velocities v at various positions (y, z) across the channel. Surface: adjustment to the theoretical expression of the velocity field of a viscous fluid in a rectangular channel (Eq 1). The color scale denotes velocities (see v axis). Black lines: distance between the experimentally-measured velocities and the adjusted values at the same location. Both the width w and the effective position y₀ of the no-slip boundary condition in the y direction were adjusted, yielding w = 0.57 mm and y₀ = 0.23 mm. The volumetric flow rate Q was set to its experimentally-imposed nominal value 1 mL/h, which yields a good adjustment in the biofilm-free case (S3 Fig). The coefficient of determination of the fit is R² = 0.92. Picture: Growing biofilm snapshot showing the fluid (gray)/biofilm (black) interface matching the boundaries given by the fit.

Fig 7. Biofilms under distinct fluid flow regimes exhibit exquisitely different gene expression.

(A) Principal component analysis and clustering of genes differentially expressed in cells grown under different conditions: L (lower shear stress and lower confinement regime, i.e. 1 mm-height channel), H (higher shear stress and higher confinement, i.e. 250 μm-height channel), E (planktonic cells in exponential phase), S (planktonic cells in stationary phase). The number of genes with a False Discovery Rate < 0.01 in our differential expression analysis, is indicated above arrows. (B) 3-component plot representing ln(L/E) vs. ln(L/H) where L, E and H designate mean expression level in samples L, E and H respectively. Each point corresponds to a gene. Red: genes related to anaerobic metabolism; blue: genes related to acid resistance; green: genes related to sulfate metabolism; black: others genes. All red and blue genes (except hypD) fall in the purple zone: they have a lower expression in samples E and a higher expression in samples L, expression in samples H being intermediate.

Fig 8. Biofilm grows at low O₂ level in a millifluidic channel.

Oxygen partial pressure (pO₂) is measured in the channel fluid using Ruthenium micelles fluorescence (see Materials and Methods). (A) pO₂ in empty channels at different flow rates (0; 1 and 2 ml/h), in 250 μm- (red) and 1 mm- (blue) height channels. (B) pO₂ in 250 μm- height channel at 1 ml/h, in the presence (dark red) or absence (light red) of a growing biofilm 12 hours after initiation. (C) pO₂ vs. flow rate in a 250 μm—height channel in the presence of the growing biofilm. Error bars (smaller than dots in C) represent standard deviation over two measurements.