## 3 Results
### Biofilm Development in Time for Two Inoculum Levels
#### 3.4.2 Study of the Biofilm Biomass: Optical Density of CV Assay  
The biofilm development was also studied in terms of biomass. The biomass was quantified following Section 2.4.2 and is represented in Figure 5a. In the beginning, the optical density (OD\({}_{590}\)) increases and reaches a peak value (approximately equalled to 12). Consequently, the OD\({}_{590}\) decreases until it reaches a plateau (approximately equal to 5). The plateau was reached after 32 h and 24 h for \(10^{4}\) and \(10^{7}\) CFU/mL of inoculum level, respectively. In Figure 5b, pictures of the biofilms after CV staining are displayed. Pictures 1-5 represent the development of the biofilm from time 0 to the OD\({}_{590}\) peak, around 17 h or 10 h for inoculum levels of \(10^{4}\) and \(10^{7}\) CFU/mL, respectively. As can be seen, the biofilm started to grow from the centre of the defined area. Then, it expanded to grow on the full area showing a coffee ring effect. The coffee ring border became more and more intense in colour when OD\({}_{590}\) reached the maximum value. After 24 h, the biofilm no longer displayed a difference in intensity between the ring (border) and the centre. It looked uniform for the entire surface (Figure 6b, picture 6). During the growth and development of the biofilm on the polystyrene surface of petri dishes, another phenomenon happened between approximately 15 h and 24 h for an inoculum level of \(10^{4}\) CFU/mL (7 h and 20 h for \(10^{7}\) CFU/mL): the biofilm started also to grow at the air-liquid (A-L) interface. Figure 5c demonstrates the development of the biofilm growing at the A-L interface from its  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameter** & \multicolumn{2}{c}{**Inoculum Level [CFU/mL]**} \\ \hline
**Estimated Value** & \(\mathbf{10^{4}}\) & \(\mathbf{10^{7}}\) \\ \hline \(\lambda\) [h] & 3.28 \(\pm\) 0.80 b  & 1.00 \(\pm\) 0.65 a  \\ \(\mu_{max}\) [1/h] & 0.791 \(\pm\) 0.057 b  & 0.472 \(\pm\) 0.040 a  \\ \(ln(N_{o})\) [ln(CFU/cm\({}^{2}\))] & 6.00 \(\pm\) 0.35 b  & 13.26 \(\pm\) 0.11 a  \\ \(ln(N_{max})\) [ln(CFU/cm\({}^{2}\))] & 18.14 \(\pm\) 0.18 a  & 17.94 \(\pm\) 0.07 a  \\ Population increase [ln(CFU/cm\({}^{2}\))] & 12.14 \(\pm\) 0.39 b  & 4.68 \(\pm\) 0.13 a  \\ RMSE & 0.5479 & 0.1570 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Growth parameters. Parameters obtained from the [PERSON] and [PERSON] (1994) model fits for inoculum levels of \(10^{4}\) and \(10^{7}\) CFU/mL [23]. The table displays the lag time (\(\lambda\)), maximum specific growth rate (\(\mu_{max}\)), the initial \(ln(N_{0})\) and the maximum cell density \(ln(N_{max})\), the absolute increase of population, the Root-Mean-Square-Error (RMSE) and the respective standard errors (SE). Parameters bearing different superscript (no letter in common) are significantly different (\(p\leq 0.05\)).  
Figure 4: Population density during _P. fluorescens_ biofilm development: black and blue dots are related to inoculum levels of \(10^{4}\) and \(10^{7}\) CFU/mL, respectively. [PERSON] and [PERSON] (1994) model fits follow the same colours: blue line for \(10^{4}\) and black line for \(10^{7}\) CFU/mL inoculum level [23].  
formation (Figure 5c, picture 1) to its disruption, which resulted in small biofilm fractions floating on top of the liquid (Figure 5c, picture 2-4).  
Figure 5: (**a**) Development of the _P. fluorescens_ biofilm biomass after CV staining. Optical density (OD\({}_{90}\)) variation in time obtained with \(10^{4}\) (black dots) and \(10^{7}\) (blue dots) CFU/mL inoculum levels. (**b**) Pictures of stained biofilms during its formation and development: pictures (1), (2), (3), (4) and (5) correspond to 3h, 4h30, 6h, 7h30 and 9h of growth, while (6) corresponds to 24-h-grown biofilm. (**c**) Pellicles corresponding to the biofilm formed at the air-liquid interface, after adding PBS solution. In picture (1), a whole pellicle covering the entire interface can be observed; from (2) to (4), it is destroyed in gradually smaller fractions, floating at the interface.