**3. Results and analysis**

#### **3.1 Rheological characterization of biofilms**

#### *3.1.1 Sweeps of frequency*

Data from the sweeps of frequency showed the difference in the viscoelasticity of the samples of biofilm (filled square) and the samples of sterile LB medium (unfilled circle) that were incubated for 6 days (**Figure 2**). For brevity, the term "unmodified LB" will refer to the standard LB medium without chemical alterations, while "modified LB" will refer to any of the five chemical additions to standard LB medium (glycerol, glucose, sucrose, NaCl, and AgNO3). The sweep of frequency of the biofilm showed frequency-independent, elastic modulus (G′) dominance over the viscous modulus (G″) for all of the samples, as expected for a weak gel (Figure S3, https://ir.library.oregonstate.edu/concern/defaults/ g158bp85b). The complex modulus (|*G*<sup>∗</sup><sup>|</sup> <sup>=</sup> <sup>∣</sup> <sup>√</sup> \_\_\_\_\_\_\_\_\_ *G*′ 2 + *G*′′ 2 <sup>∣</sup>) for the samples of biofilm (~0.02 Pa) were, on average, an order of magnitude larger than their counterparts of sterile LB medium (**Figure 2a**). Previous work on the sterile LB medium showed that aging and cycling of temperature can cause buildup of protein at the air-liquid interface, which can be measured by a plate geometry to an extent, though not as accurately as using a du Noüy ring. Therefore, the measurable viscoelasticity in the sterile samples can be attributed to the age of the medium at the time of use (>3 months) and to the incubation process, which further accelerated the aging. The complex modulus of the unmodified LB biofilm and the sterile unmodified LB medium were each averaged over the entire frequency range (0.1–1 rad s<sup>−</sup><sup>1</sup> ) and were averaged between replicates (biofilm n = 12, sterile n = 7) to get mean values for G\*. All the raw results from sweeps of frequency of the biofilm and of the sterile samples in modified LB medium (Figure S2, https:// ir.library.oregonstate.edu/concern/defaults/g158bp85b) were averaged over the entire frequency range and plotted (Figure S3b–f, https://ir.library.oregonstate. edu/concern/defaults/g158bp85b). The mean values of G\* for biofilms that were grown in unmodified LB (red) served as the base of comparison for biofilms that were grown in modified LB medium (**Figure 2b**–**f**). Based on the modulus data, bacterial biofilm appeared to be strongly affected by its nutritional environment.

#### *3.1.2 Sweeps of stress*

The values of yield stress were derived from the experiments of increasing strain, where the yield stress is the point of offset of a stress-versus-strain curve.

**13**

sweeps of frequency.

**Figure 2.**

*3.1.3 Modification with glycerol*

well as inducing high osmolarity (1.4 Osm L<sup>−</sup><sup>1</sup>

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns…*

The stress-versus-strain data of the unmodified LB biofilm (filled square) and sterile unmodified LB samples (unfilled circle) showed that the samples of biofilm exhibited an appreciable yield stress (**Figure 3a**). After yielding, the material stress was constant (<1 Pa) at high strain, while the samples of sterile medium demonstrated no yield stress. In fact, none of the sterile modified or unmodified LB mediums had an appreciable yield stress (Figure S2, https://ir.library.oregonstate.edu/ concern/defaults/g158bp85b). The mean yield stress of the samples of unmodified LB biofilm (red) was compared with the biofilms that were grown on the modified LB medium (**Figure 3b**–**f**). The results of yield stress showed a similar dependence on the nutritional conditions of the biofilms as the results of the modulus from the

*and (f) represent inhibiting concentrations that had no biofilm growth.*

*Modulus |G\*| calculated from the sweep of frequency showing the biofilm (filled square) and sterile LB medium (unfilled circle) samples. The results of the (a) sweep of frequency from ω* ∈*[0.1, 1] rad s<sup>−</sup><sup>1</sup>*

*γo = 0.1 and sterile LB medium γo = 0.005) of biofilm samples that were grown in unmodified LB medium and of sterile unmodified LB medium. The mean modulus |G\*| was 0.015 Pa for biofilm and 0.0015 Pa for sterile unmodified LB medium. (b–f) The mean modulus |G\*| is plotted (red) with relative errors that were calculated from standard error to ensure symmetry (n* ≥ *3). The average |G\*| of biofilm that was grown in modified LB medium with 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, (d) 1.5–5 w/v% NaCl and (e) 0.001–1 mM AgNO3 are shown. The gray regions in (b)* 

With the addition of glycerol (0–15 v/v%), the complex modulus of the biofilm increased by almost an order of magnitude between 0 and 2% glycerol, remained constant between 2 and 10%, and experienced a dramatic drop in modulus for concentrations greater than 10% to modulus values that are comparable to sterile LB (**Figure 2b**). The yield stress of the biofilm showed similar trends, increasing by one order of magnitude with glycerol from 0 to 10% until concentrations of glycerol that were greater than 10% impeded the growth of biofilm, also resulting in no yield stress (**Figure 3b**). The addition of glycerol increased the viscosity of the medium as

Other studies with glycerol-supplemented medium saw an increase in the production of EPS by biofilm, consistent with the present study [27, 70]. The glycerol can trigger

at 10%), promoting stronger biofilm.

 *(biofilm* 

*DOI: http://dx.doi.org/10.5772/intechopen.85240*

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns… DOI: http://dx.doi.org/10.5772/intechopen.85240*

#### **Figure 2.**

*Pseudomonas aeruginosa - An Armory Within*

**2.4 Dried bacterial biofilms under the microscope**

images were produced with polarized filters.

**3.1 Rheological characterization of biofilms**

g158bp85b). The complex modulus (|*G*<sup>∗</sup><sup>|</sup> <sup>=</sup> <sup>∣</sup> <sup>√</sup>

**3. Results and analysis**

*3.1.1 Sweeps of frequency*

range (0.1–1 rad s<sup>−</sup><sup>1</sup>

*3.1.2 Sweeps of stress*

the photographs to black and white image on MATLAB (Figure S4, https://ir.library. oregonstate.edu/concern/defaults/g158bp85b), and finally calculating the ferning coverage by determining the ratio between white and black pixel areas in the image. The fractal dimension was calculated using the box-counting method on MATLAB

Microscopic images of the biofilm in its liquid and its dried ferning state were taken using an Eclipse Ti-S inverted microscope (Nikon, Japan). The polarized

Data from the sweeps of frequency showed the difference in the viscoelasticity of the samples of biofilm (filled square) and the samples of sterile LB medium (unfilled circle) that were incubated for 6 days (**Figure 2**). For brevity, the term "unmodified LB" will refer to the standard LB medium without chemical alterations, while "modified LB" will refer to any of the five chemical additions to standard LB medium (glycerol, glucose, sucrose, NaCl, and AgNO3). The sweep of frequency of the biofilm showed frequency-independent, elastic modulus (G′) dominance over the viscous modulus (G″) for all of the samples, as expected for a weak gel (Figure S3, https://ir.library.oregonstate.edu/concern/defaults/

\_\_\_\_\_\_\_\_\_ *G*′ 2 + *G*′′ 2

) and were averaged between replicates (biofilm n = 12, sterile

(~0.02 Pa) were, on average, an order of magnitude larger than their counterparts of sterile LB medium (**Figure 2a**). Previous work on the sterile LB medium showed that aging and cycling of temperature can cause buildup of protein at the air-liquid interface, which can be measured by a plate geometry to an extent, though not as accurately as using a du Noüy ring. Therefore, the measurable viscoelasticity in the sterile samples can be attributed to the age of the medium at the time of use (>3 months) and to the incubation process, which further accelerated the aging. The complex modulus of the unmodified LB biofilm and the sterile unmodified LB medium were each averaged over the entire frequency

n = 7) to get mean values for G\*. All the raw results from sweeps of frequency of the biofilm and of the sterile samples in modified LB medium (Figure S2, https:// ir.library.oregonstate.edu/concern/defaults/g158bp85b) were averaged over the entire frequency range and plotted (Figure S3b–f, https://ir.library.oregonstate. edu/concern/defaults/g158bp85b). The mean values of G\* for biofilms that were grown in unmodified LB (red) served as the base of comparison for biofilms that were grown in modified LB medium (**Figure 2b**–**f**). Based on the modulus data, bacterial biofilm appeared to be strongly affected by its nutritional environment.

The values of yield stress were derived from the experiments of increasing strain, where the yield stress is the point of offset of a stress-versus-strain curve.

<sup>∣</sup>) for the samples of biofilm

(Figure S4, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b).

**12**

*Modulus |G\*| calculated from the sweep of frequency showing the biofilm (filled square) and sterile LB medium (unfilled circle) samples. The results of the (a) sweep of frequency from ω* ∈*[0.1, 1] rad s<sup>−</sup><sup>1</sup> (biofilm γo = 0.1 and sterile LB medium γo = 0.005) of biofilm samples that were grown in unmodified LB medium and of sterile unmodified LB medium. The mean modulus |G\*| was 0.015 Pa for biofilm and 0.0015 Pa for sterile unmodified LB medium. (b–f) The mean modulus |G\*| is plotted (red) with relative errors that were calculated from standard error to ensure symmetry (n* ≥ *3). The average |G\*| of biofilm that was grown in modified LB medium with 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, (d) 1.5–5 w/v% NaCl and (e) 0.001–1 mM AgNO3 are shown. The gray regions in (b) and (f) represent inhibiting concentrations that had no biofilm growth.*

The stress-versus-strain data of the unmodified LB biofilm (filled square) and sterile unmodified LB samples (unfilled circle) showed that the samples of biofilm exhibited an appreciable yield stress (**Figure 3a**). After yielding, the material stress was constant (<1 Pa) at high strain, while the samples of sterile medium demonstrated no yield stress. In fact, none of the sterile modified or unmodified LB mediums had an appreciable yield stress (Figure S2, https://ir.library.oregonstate.edu/ concern/defaults/g158bp85b). The mean yield stress of the samples of unmodified LB biofilm (red) was compared with the biofilms that were grown on the modified LB medium (**Figure 3b**–**f**). The results of yield stress showed a similar dependence on the nutritional conditions of the biofilms as the results of the modulus from the sweeps of frequency.

### *3.1.3 Modification with glycerol*

With the addition of glycerol (0–15 v/v%), the complex modulus of the biofilm increased by almost an order of magnitude between 0 and 2% glycerol, remained constant between 2 and 10%, and experienced a dramatic drop in modulus for concentrations greater than 10% to modulus values that are comparable to sterile LB (**Figure 2b**). The yield stress of the biofilm showed similar trends, increasing by one order of magnitude with glycerol from 0 to 10% until concentrations of glycerol that were greater than 10% impeded the growth of biofilm, also resulting in no yield stress (**Figure 3b**). The addition of glycerol increased the viscosity of the medium as well as inducing high osmolarity (1.4 Osm L<sup>−</sup><sup>1</sup> at 10%), promoting stronger biofilm. Other studies with glycerol-supplemented medium saw an increase in the production of EPS by biofilm, consistent with the present study [27, 70]. The glycerol can trigger

#### **Figure 3.**

*(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) represent inhibiting concentrations that had no biofilm growth.*

pathways of production of EPS; [27] however, at high concentrations of glycerol, the diffusion-limiting environment of the highly viscous solution with high osmotic pressure (>4 Osm L<sup>−</sup><sup>1</sup> at >10%) appeared to inhibit growth. The complex modulus of 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, which is a bluish-tinted toxin that is produced by PAO1.

#### *3.1.4 Modification with glucose*

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> , which did not cause inhibiting effects.

**15**

[34, 71].

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns…*

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>

the present work, samples of PAO1 experienced a maximum of 0.13 Osm L<sup>−</sup><sup>1</sup>

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

Unmodified LB medium already consists of sodium chloride (NaCl) at a concentration of 1%, and the modified concentration varied from 1 to 5% of NaCl. The complex modulus of biofilm remained constant for concentrations below 2.5% and increased by one order of magnitude for concentrations between 2.5 and 5%, while the modulus of the sterile modified LB medium was not affected by the concentration of NaCl (**Figure 2e**). Similarly, the yield stress increased as the concentration of NaCl was increased greater than 2.5% (**Figure 3e**). NaCl is already required for bacterial growth to provide osmotic balance, but a larger amount of salts appeared to promote stronger biofilm. The change in the biofilm could be caused by the higher salinity or osmolarity, making the environment hostile, triggering a higher level of production of alginate and other types of EPS as a countermeasure. Previous studies found that concentrations of NaCl between about 1 and 3%

increased production of biofilms in *S. aureus* and *P. aeruginosa*, while concentrations of about 6% of NaCl prevented growth of biofilm in *S. aureus* [29, 31]. At concentrations of NaCl above 10%, no biofilm growth was observed, and the plate quickly crystalized to cubes of salt (Figure S6, https://ir.library.oregonstate.edu/concern/

Silver has antimicrobial properties that can inhibit bacterial growth and development of biofilm. Supplementation of silver nitrate (AgNO3) to the modified LB medium has no impact on the complex modulus (**Figure 2f**) or the yield stress of the biofilm for concentrations below 0.1 mM (**Figure 3f**). Past this concentration, the modulus instantly reduced to the same level as the sterile modified LB medium, and the yield stress disappeared. Correspondingly, the plates of biofilm at the higher silver concentrations appeared clear and less viscous, resembling sterile modified LB medium (Figure S1, https://ir.library. oregonstate.edu/concern/defaults/g158bp85b). Therefore, the antimicrobial activity of the silver appeared to be strongly dependent on concentration, with little to no effect on bacterial growth at concentrations lower than the threshold (0.1 mM) and deadly at higher concentrations. These results are consistent with previous studies where the inhibitory threshold for *S. aureus* was over 0.033 mM,

while the inhibitory threshold for *P. aeruginosa* was over 0.16 μg mL<sup>−</sup><sup>1</sup>

) [30]. In

(0.45 mM)

in

*DOI: http://dx.doi.org/10.5772/intechopen.85240*

*3.1.5 Modification with sucrose*

properties of the biofilm.

defaults/g158bp85b).

*3.1.7 Modification with silver nitrate*

*3.1.6 Modification with sodium chloride*

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns… DOI: http://dx.doi.org/10.5772/intechopen.85240*
