Effects of fluid flow conditions on interactions between species in biofilms

Abstract

Most microorganisms live in complex communities, where they interact both synergistically and competitively. To explore the relationship between environmental heterogeneity and the spatial structure of well-defined biofilms, single- and mixed-species biofilms of Pseudomonas aeruginosa PAO1 and Flavobacterium sp. CDC-65 was grown in a planar flow cell under highly controlled flow gradients. Both organisms behaved differently in mixed cultures than in single-species cultures due to inter-species interactions, and these interactions were significantly affected by external flow conditions. Pseudomonas and Flavobacterium showed a competitive relationship under slow inflow conditions, where the supply of growth medium was limited. Under such competitive conditions, the faster- specific growth rate of Flavobacterium allowed it to secure access to favorable regions of the biofilm by overgrowing Pseudomonas. In contrast, Pseudomonas was restricted to nutritionally depleted habitat near the base of the biofilm, and its growth was significantly inhibited. Conversely, under higher inflow conditions providing greater influx of growth medium, both organisms accumulated greater biomass in mixed biofilms than in single-species biofilms. Spatial segregation of the two organisms within the biofilms contributed to enhanced overall exploitation of available nutrients and substrates, while morphological changes favored better adherence to the surface under high hydrodynamic shear. These results indicate that synergy and competition in biofilms vary with flow conditions. Limited resource replenishment favors competition under low-flow conditions, while high flow reduces competition and favors synergy by providing greater resources and simultaneously imposing increased hydrodynamic shear that makes it more difficult to accumulate biomass on the surface. Ecological interactions that produce mechanically stronger and more robust biofilms will support more extensive growth on surfaces subject to high hydrodynamic shear, but these interactions are difficult to predict from observations of the behavior of individual organisms.

Figure 1

A) Photograph of the planar flow cell. B) The flow field in the flow chamber with streamlines under inflow rate of 0.16 mL min⁻¹. C) The observation window in the center of the flow chamber, including locations of nine regions observed by confocal microscopy. Coordinates are lattice locations normalized by lattice spacing (Zhang, et al., 2011), and color bars of U represent lattice dimensionless fluid velocity increased from blue to red.

Figure 2

Dual-species seven-day-old biofilms of Pseudomonas aeruginosa PAO1 (green) and Flavobacterium sp. CDC-65 (red) under inflow rates of 0.16 mL min⁻¹ and 0.8 mL min⁻¹. A) and B) show the representative biofilm morphology on the 7^th day at each of the nine regions indicated in Figure 1C. Local velocities in each region are given in parentheses, and the grid unit is 37.6 μm. C) and D) show the representative vertical distributions of Pseudomonas and Flavobacterium at each region of the biofilm. Red lines represent the fraction of Flavobacterium and green lines represent the fraction of Pseudomonas.

Figure 3

Representative seven-day-old single-species biofilms of Pseudomonas aeruginosa PAO1 and Flavobacterium sp. CDC-65 under inflow rates 0.16 mL min⁻¹ and 0.80 mL min⁻¹. Results are shown for each of the nine regions indicated in Figure 1C. Local velocities in each region are given in parentheses, and the grid unit is 37.6 μm.

Figure 4

Biofilm biomass and morphology as a function of local velocity in single- and mixed-species biofilms. Open symbols represent single-species biofilms of Pseudomonas aeruginosa PAO1 and Flavobacterium sp. CDC-65, and solid symbols represent mixed biofilms. A) Total biomass of single- and mixed-species biofilms, and biomass of each organism within mixed biofilms. B) Average thickness, C) Roughness and D) Surface-area-to-volume ratio (As/V) of single- and mixed-species biofilms. Plotted points and error bars represent the average and standard deviation of three replicate observations. Similar trends were observed in other experiments.

Figure 5

Relationship between biofilm biomass and solute travel time from the flow-cell inlet to the observation locations. Open symbols represent single-species biofilms, and solid symbols represent mixed biofilms. Black icons show observations obtained under fast inflow rate (0.80 mL min⁻¹), and gray icons in the inset show observations obtained under the slow inflow rate (0.16 mL min⁻¹). Plotted points and error bars represent the average and standard deviation of three replicate observations. Similar trends were observed in other experiments.