*3.1.5 Modification with sucrose*

*Pseudomonas aeruginosa - An Armory Within*

pathways of production of EPS; [27] however, at high concentrations of glycerol, the diffusion-limiting environment of the highly viscous solution with high osmotic

*(a) The stress versus strain data of biofilm that was grown in unmodified LB medium (filled square) and of samples of sterile unmodified LB medium (unfilled circle). (b–f) For the mean yield stress of biofilm that was grown in unmodified LB medium (red square), the standard error bars were converted to relative errors to ensure symmetry on the y-axis (n* ≥ *7). Plots of yield stress τy of the biofilms that were grown in modified LB medium at the following concentrations (b) 1–15 v/v% glycerol, (c) 0.5–4.5 w/v% glucose, (d) 0.5–4.5 w/v% sucrose, (e) 1.5–5 w/v% NaCl and (f) 0.001–1 mM AgNO3 are shown. The mean τy was 0.32 Pa for the unmodified LB biofilms, while the LB medium did not have a yield stress. The gray regions in (b) and (f)* 

which is a bluish-tinted toxin that is produced by PAO1.

*represent inhibiting concentrations that had no biofilm growth.*

the modified LB medium, on the other hand, stayed relatively constant with glycerol addition. The dramatic drop in the modulus of biofilm samples that were grown in medium that was modified with >10% glycerol corresponded with an apparent lack of biofilm in the Petri dishes, as the dishes appeared clear and yellow instead of opaque and greenish (Figure S1, https://ir.library.oregonstate.edu/concern/defaults/ g158bp85b). The greenish hue in the samples is a result of the presence of pyocyanin,

The modulus of the biofilm increased by one order of magnitude by increasing the concentration of glucose from 0 to 4.5% (**Figure 2c**), indicating that glucose was being utilized by the bacteria as an additional source of carbon which promoted growth and development of a stronger network of biofilm. The rheological results of the sterile glucose-modified LB medium did not change significantly from the unmodified LB medium. The values of yield stress followed the same trend, where the biofilm that was grown in glucose-modified LB medium had yield stresses that were an order of magnitude larger than the unmodified LB biofilm (**Figure 3c**). A previous study observed the same effect, finding that the addition of glucose up to the highest level tested, which was 2.7%, enhanced biofilm production [29]. The maximum addition of glucose (4.5%) induced osmotic pressure of 0.25 Osm L<sup>−</sup><sup>1</sup>

at >10%) appeared to inhibit growth. The complex modulus of

,

**14**

pressure (>4 Osm L<sup>−</sup><sup>1</sup>

**Figure 3.**

*3.1.4 Modification with glucose*

which did not cause inhibiting effects.

Based on the rheology, sucrose did not increase biofilm production, as no change existed in the modulus (**Figure 2d**) or yield stress (**Figure 3d**) of the biofilm. In previous studies, concentrations of sucrose above 10% in medium for *P. aeruginosa* resulted in biofilm with mucoid development, while *P. fluorescens* started to experience adverse effects above 15% at which point the biofilm dramatically decreased [30, 31]. In those studies, bacterial culture reached an inhibiting level of sucrose at 15% due to osmotic pressure (0.44 Osm L<sup>−</sup><sup>1</sup> ) [30]. In the present work, samples of PAO1 experienced a maximum of 0.13 Osm L<sup>−</sup><sup>1</sup> in osmotic pressure from modification with sucrose, which is well below the reported osmotic level for inhibition. *P. aeruginosa* may not be capable of utilizing sucrose, so in contrast to the simpler glucose, sucrose had little impact on the rheological properties of the biofilm.
