## 4 Discussion
### Biofilm Dynamics for Two Inoculum Levels
#### 4.2.1 Biofilm Population Density: Growth Parameters
Since both biofilms have the same initial concentration of nutrients available in their growth medium, the biofilm inoculated at \(10^{4}\) CFU/mL will have a lower nutrient concentration when reachingthe initial concentration of the biofilm that was inoculated at \(10^{7}\) CFU/mL. The growth rate is expected to decrease with the nutrient concentration in the given range. As such, it is unlikely that nutrient availability can explain the faster growth rate of the biofilm with the inoculum level of \(10^{4}\) CFU/mL. The same reasoning could be applied to the potential presence of toxic cell metabolites. A study of [PERSON] et al. demonstrated that biofilms growing under flow conditions developed as mushroom structures at low inoculum levels whereas they developed as a layer of connected colonies at very high inoculum levels [58]. This research was conducted using an agent-based model that was validated using micrographs of the real biofilms that were formed in channel \(\upmu\)-Slides under flow conditions. The authors underlined that the competition between the motile and immotile subpopulations also played a fundamental role [58]. The model of [PERSON] and [PERSON] (1994) is a simple empirical model which, in this specific case, is not able to explain the mechanism behind the differences between the relative maximum growth rates as a result of the different inoculum levels [23]. A dedicated model for biofilms should take into account the complexity of the system since the model in its current form cannot consider different complex phenomena occurring, e.g., adhesion of new cells, trapping of cells, cell detachment or death, cell division [50; 56]. All these processes might influence the cell population and its increase within the biofilms. Finally, the two growth curves reached the same maximum cell density during the stationary phase (plateau). The reason of the plateau was due to the nutrient depletion in the drop, which was similar to the behaviour observed in planktonic form [59]. Other authors, using a modified Gompertz model, investigated the effect of different temperatures and pH on _Salmonella_ Typhimurium biofilm growth, and the population density reached the same value, as well. Despite using an inoculum level of \(10^{4}\) CFU/mL, a population density of 18 ln(CFU/cm\({}^{2}\)) using a similar static method was obtained. The difference between their protocol and the current research is related to the use of coupon method [60]. [PERSON] et al. demonstrated that _P. fluorescens_ and _P. gismontii_ followed the [PERSON] and [PERSON] model and reached a stationary phase, regardless of the method used for the biofilm growth, i.e., static (hydrodynamic) in petri dishes or dynamic using other biofilm-developing-methods [23; 56]. [PERSON] and [PERSON], who investigated the biofilm cell response to food-related stress, obtained _P. fluorescens_ biofilms on coupons (static hydrodynamic method) starting with an inoculum concentration of \(10^{6}\) CFU/mL [35]. Although the authors grew the biofilms for 48 h at 10 \({}^{\circ}\)C, they obtained a biofilm equally dense to the one developed in the present research, corresponding to about 7.5 log (CFU/cm\({}^{2}\)) (corresponding to 17.3 ln (CFU/cm\({}^{2}\)). Finally, [PERSON] et al. also, using a different biofilm-developing-method (flow cell system), underlined that _P. fluorescens_ biofilms reach their steady state between 12 and 60 h, and did not further increase [39]. These studies confirm the reliability of the developed protocol, as the typical growth pattern, i.e., reaching a stationary phase with a dense biofilm (irrespective to the specific inoculum conditions), was reproduced using the novel method proposed.