Mixed-species biofilms under flow gradients
After 5 days of growth, the system reached a steady state in which the overall pattern of biofilm morphology and the distribution of the two species within the biofilm no longer changed with time. Figure 2 shows the biofilm morphology and spatial distributions of each organism within 7-day-old mixed-species biofilms.
Under slow inflow conditions, thin biofilms composed of both Pseudomonas and Flavobacterium were detected in all regions. Pseudomonas was present as sporadic small colonies and single cells, while Flavobacterium formed relatively continuous biofilms with larger clusters (Fig. 2a). Regions R1, R2, and R4 had thinner Flavobacterium biofilms and more Pseudomonas colonies. Vertical distributions, presented in Fig. 2c, indicate that Pseudomonas mainly occurred at the base of mixed biofilms and was overgrown by Flavobacterium. In all regions, at all elevations, Flavobacterium was the dominant component of biomass in the 7-day biofilm.
Under fast inflow conditions, more complex intergrowth of the two organisms was observed. The biofilm morphologies and the vertical distributions of each organism are shown in Fig. 2b and d, respectively. Pseudomonas always formed the base of mixed biofilms, while Flavobacterium dominated the upper part of the biofilm canopy. In regions R1, R4, and R7, Pseudomonas occurred in a smooth, continuous, and thin base layer, while Flavobacterium formed discontinuous large tufts and clusters on top of Pseudomonas biofilms. In these regions, Pseudomonas was the dominant organism at the base of the biofilm: it represented over 50% of the biomass in the bottom 10 μm, and Flavobacterium was the dominant organism above 10 μm. The fraction of Pseudomonas gradually decreased until it was absent at a height of 27 μm in R1 and at 16 μm in R4 and R7. The maximum height of the Flavobacterium biofilm was 36 μm. In locations R2 and R5, Flavobacterium also existed as large tufts with pillars and towers of cells, while Pseudomonas occurred only in a thin and continuous layer at the base of the biofilm. In these regions, Flavobacterium reached maximum thicknesses of 50 μm at location R2 and 58 μm at location R5, while Pseudomonas disappeared at 19 μm (R2) and 15 μm (R5). Smoother biofilms formed in regions of faster local velocity. Region R8 had a faster velocity (9.78 cm min−1), leading to greater accumulation of Pseudomonas and Flavobacterium and the formation of some tower structures. Pseudomonas was the dominant organism to a height of 18 μm and disappeared at 25 μm, while Flavobacterium was found at a maximum height of 44 μm. Regions R6 and R9 had the highest velocities (10.1 and 10.4 cm min−1, respectively) and accumulated more Pseudomonas than other regions. Pseudomonas was the dominant organism to a height of 23 μm at R6 and 16 μm at R9, whereas Flavobacterium only achieved maximum heights of 36 μm at R6 and 29 μm at R9.
Single-species biofilms under flow gradients
We observed the behavior of single-species Pseudomonas and Flavobacterium biofilms to provide a basis for evaluating their interactions in mixed-species biofilms. Single-species Pseudomonas biofilms achieved steady state after 4 days, and single-species Flavobacterium biofilms achieved steady state after 6 days. Figure 3 shows the morphologies of 7-day single-species biofilms of Pseudomonas and Flavobacterium under the two inflow rates.
Figure 3. Representative 7-day-old single-species biofilms of Pseudomonas aeruginosa PAO1 and Flavobacterium sp. CDC-65 under inflow rates 0.16 and 0.80 mL min−1. Results are shown for each of the nine regions indicated in Fig. 1c. Local velocities in each region are given in parentheses, and the grid unit is 37.6 μm.
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Morphological gradients were observed in single-species Pseudomonas biofilms relative to the imposed flow gradient. Pseudomonas formed sporadic small colonies under the slower inflow condition. When the local velocity increased from 2.52 to 3.72 cm min−1, the number of colonies increased with local flow velocity, but there was no continuous biofilm detected under these conditions (Fig. 3a). More continuous biofilms formed under the faster inflow condition (Fig. 3c). Large clusters formed in region R1, where the local velocity was 5.76 cm min−1. In regions R2 and R4, which had higher velocities of 7.62 and 7.50 cm min−1, respectively, clusters connected together leading to higher surface coverage. Smooth, continuous biofilms developed in region R7, where the local velocity was 9.00 cm min−1. Additional roughness developed under higher velocities at R5 and R3, and then more clusters, towers, and mushroom features appeared under the fastest velocities in regions R6, R8, and R9.
Single-species Flavobacterium biofilms developed different morphologies under the same flow conditions. Under the slow inflow rate, very thin and discontinuous biofilms formed, and there was no significant difference between regions R1–R9, as shown in Fig. 3b. Loose and filamentous biofilms formed under the fast inflow rate, and biofilm structures changed with local velocities as shown in Fig. 3d. Sparse biofilms formed in region R1 under the flow velocity 5.76 cm min−1. Rougher and thicker biofilms formed in regions R2, R3, R4, R5, and R7, which had higher velocities ranging from 7.50 to 9.36 cm min−1. When the local velocity exceeded 9.78 cm min−1, biofilms became looser and were unable to fully cover the substratum (regions R6, R8, and R9).
Quantitative description of biofilm morphology
Quantitative analysis of the confocal images supports more specific comparisons of differences in biofilm structure with flow conditions. We quantitatively compared the biofilm morphology using four parameters calculated from the confocal images: biomass per unit area (μm3 μm−2), average biofilm thickness (μm), roughness (dimensionless), and surface-area-to-volume ratio (As/V, μm2 μm−3) (Heydorn et al., 2000a, b). These parameters are plotted as functions of local velocity in Fig. 4.
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.
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Under velocities of 2.52–3.72 cm min−1, Flavobacterium in mixed biofilms accumulated 0.4–0.9 μm3 μm−2 biomass, while Pseudomonas biomass only reached 0.11–0.22 μm3 μm−2. Overall, Flavobacterium accumulated 4–7 times more biomass than Pseudomonas and was the dominant member of the consortium in mixed biofilms (P < 0.01). The average thickness of the mixed biofilms increased from 0.34 to 0.74 μm under velocities of 2.52–3.24 cm min−1 and then reached a constant value of 0.73 μm under velocities of 3.24–3.72 cm min−1.
Under the fast-flow conditions, the biomass of Pseudomonas in mixed cultures was around 7.0 μm3 μm−2 under velocities of 7.50–9.00 cm min−1 and rose above 10.0 μm3 μm−2 when velocity exceeded 9.36 cm min−1. A positive relationship was observed between biomass accumulation and local velocity for Flavobacterium under low velocities, but this trend reversed under high local velocity. The Flavobacterium biomass increased from 3.8 μm3 μm−2 at 5.76 cm min−1 to 15.1 μm3 μm−2 at 9.78 cm min−1, but declined to 12.6 μm3 μm−2 at 10.1 cm min−1 and even further to 9.2 μm3 μm−2 at 10.4 cm min−1 (R9). Total biomass showed the same trend as Flavobacterium, with a positive relationship between biomass accumulation and velocity until the velocity exceeded 9.78 cm min−1. The average thickness of the mixed biofilms reached 38.9 μm under a velocity 9.78 cm min−1 and then decreased with velocity to 32.7 μm under 10.1 cm min−1 and 26.5 μm under 10.4 cm min−1 (Fig. 4b).
The roughness of mixed biofilms showed an inverse relationship with velocity under slow inflow conditions, with roughness decreasing from 1.0 to 0.7 as velocity increased from 2.52 to 3.72 cm min−1. Under fast-flow conditions, the roughness of mixed biofilms varied between 0.1 and 0.3 (Fig. 4c). As/V values of mixed biofilms ranged from 1.5 to 2.8 μm2 μm−3 under velocities of 2.52–10.4 cm min−1. In addition, mixed biofilms with more Pseudomonas biomass had less complex structures than those dominated by Flavobacterium. For instance, the lowest As/V observed in mixed biofilms, 1.5, occurred in locations R1 and R3 under velocities of 5.76 and 9.36 cm min−1, where Pseudomonas had significantly greater biomass than Flavobacterium (Fig. 4a).
To compare the difference between single-species biofilms and mixed-species biofilms, characteristics of single-species biofilms are shown in Fig. 4. Under slow inflow conditions, biomass of both single-species biofilms showed a positive relationship with local velocity (inserts in Fig. 4a and b). Single-species Flavobacterium biofilms had greater biomass (0.06–1.18 μm3 μm−2) than single-species Pseudomonas biofilms (biomass 0.03–0.46 μm3 μm−2). Compared with mixed biofilms, Pseudomonas accumulated more biomass in single-species cultures (P < 0.01), but Flavobacterium did not show a significant difference between mixed and single-species cultures (P > 0.05).
Under fast inflow conditions, single-species Pseudomonas biofilms showed a slight increase from 4.4 to 7.5 μm3 μm−2 over the velocity range of 5.76–10.4 cm min−1 and then maintained around 7.0 μm3 μm−2 of biomass up to a velocity of 10.4 cm min−1. Single-species Flavobacterium biofilms accumulated less biomass than single-species Pseudomonas: 0.99 μm3 μm−2 under 5.76 cm min−1 and 1.6–2.6 μm3 μm−2 at velocities from 7.50 to 10.4 cm min−1. In addition, Flavobacterium accumulated significantly less biomass in single-species biofilms than it did in mixed biofilms (P < 0.001). Under higher local velocities, the average thickness of single-species Pseudomonas remained at 4.6–10.3 μm, while the thickness of single-species Flavobacterium increased slightly with velocity, though less than observed for single-species Pseudomonas. The single-species biofilms achieved significantly lower average thicknesses everywhere than mixed biofilms: single-species Pseudomonas biofilms were 2–4 times thinner, and single-species Flavobacterium biofilms were 3–10 times thinner than the mixed-species biofilm (Fig. 4b).
When local velocities increased from 2.52 to 3.72 cm min−1, the roughness of both single-species biofilms declined from 2.0 to 1.7 with Pseudomonas and from 2.0 to 1.6 with Flavobacterium. Similar behavior was observed in mixed biofilms, but single-species biofilms were rougher under all conditions. Under higher velocities, 5.76–10.4 cm min−1, single-species Pseudomonas biofilms maintained a low roughness of 0.2–0.4, but Flavobacterium biofilms had a high roughness of 1.2–1.5 (Fig. 4c).
In Fig. 4d, it can be seen that As/V values of single-species Pseudomonas ranged between 2.0 and 3.1 μm2 μm−3 under slow inflow conditions, but single-species Flavobacterium had much greater As/V values of 5.0–8.2 μm2 μm−3. With the greater velocity under the fast inflow conditions, the As/V values of both organisms decreased. Single-species Pseudomonas biofilms had a small As/V of 0.5–1.5 μm2 μm−3 under velocities of 5.76–10.4 cm min−1, and single-species Flavobacterium biofilms had As/V ranging from 3.7 to 4.8 μm2 μm−3. As/V values of mixed biofilms did not significantly change from slow inflow conditions to fast inflow conditions. Moreover, they were similar with the values of single-species Pseudomonas biofilms under slow inflow conditions, while they were greater than that of single-species Pseudomonas biofilms and smaller than that of single-species Flavobacterium biofilms under fast inflow conditions.
Effects of solute transport on biofilm growth
Energy-yielding substrates and nutrients in growth media are consumed by metabolically active cells within biofilms. To test the effects of this depletion, we evaluated biofilm properties as a function of solute travel time and distance from the flow cell inlets. The travel distance to each location within the flow cell was calculated by integrating along the flow paths determined using the lattice Boltzmann model simulations, and the travel time was calculated similarly by integrating the local velocity along flow paths. Travel time and distance here are surrogate measures for the overall depletion of the growth medium during transport through the flow cell. We used these measures because we could calculate them directly for all locations in the flow cell, but we could not measure relevant chemical concentrations in situ. The solute travel time and distance from the flow cell inlets were calculated by the lattice Boltzmann simulations based on the velocity field shown in Fig. 1b (Zhang et al., 2011). The relationship between biomass accumulation and solute travel time is shown in Fig. 5, and the associated relationship with travel distance is presented in Supporting Information, Fig. S1.
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.
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In single-species Pseudomonas biofilms, an inverse relationship was found between the solute travel time and biomass: when solute travel time increased from 0.6 to 8.7 min, Pseudomonas biomass decreased from 7.5 to 0.03 μm3 μm−2. A similar but weaker trend was found for Pseudomonas in mixed biofilms, with accumulated biomass decreasing from 12.2 μm3 μm−2 at 0.6 min to 0.11 μm3 μm−2 at 8.7 min. Single-species Flavobacterium biofilms accumulated biomass of 0.06–2.7 μm3 μm−2, but the amount of biomass did not decrease with solute travel time. Flavobacterium accumulated more biomass in mixed biofilms than in single-species culture, and interestingly, in mixed biofilms it also showed the inverse relationship between biomass and solute travel time as that observed for Pseudomonas. The biomass of Flavobacterium in mixed biofilms decreased from 12.3 to 0.43 μm3 μm−2 as solute travel time increased from 0.6 to 8.7 min. Total biomass of mixed biofilms also showed a significant inverse relationship over the same range of solute travel time, with observed total biomass decreasing from 27.3 to 1.27 μm3 μm−2.
We will now consider the inverse relationship between biofilm biomass and travel time for the mixed culture in more detail. Under the fast inflow condition, regions R2 and R4 had similar velocities and shear stresses, but biomass abundance in R2 was 19.1 μm3 μm−2, greater than that observed at R4 (17.0 μm3 μm−2). This difference is explained by the difference in the solute travel time to these regions. The travel time to R2 was much faster than to R4 (1.1 min vs. 1.8 min). Regions R3, R5, and R7 also showed similar local velocities but inverse relationships between travel time and biomass (travel times: R3 = 0.8 min, R5 = 1.3 min, R7 = 2.1 min, biomass accumulation: R3 = 20.6 μm3 μm−2, R5 = 19.7 μm3 μm−2 and R7 = 15.8 μm3 μm−2). The only exception to this trend was region R8 under the fast-flow condition, where the travel time was 1.1 min but the accumulated biomass was 29.4 μm3 μm−2. No clear relationship was found between biomass accumulation and solute travel distance in either single- or mixed-species biofilms under either flow condition (Fig. S1). Note that the fluid velocity varies substantially within the flow cell (Fig. 1), so the solute travel time is not linearly proportional to the solute travel distance. Therefore, these results indicate that the travel time, and not simply travel distance, provides the best measure of the consumption of growth medium from community metabolism along flow paths.