## 4 Discussion
### Biofilm Dynamics for Two Inoculum Levels
#### 4.2.2 Biofilm Biomass: Optical Density Evolution and Pellicles  
An interesting result was found when the development of the biomass was investigated. Our findings did not support previous research in this field. In fact, the optical density did not follow the [PERSON] and [PERSON] (1994) model, as previous research indicated for biofilms of different strains [23; 44]. This means that the total biomass as a function of time is not simply proportional to the biofilm cell density. In the current study, the optical density reached a maximum value and then decreased. It is important to divide the biofilm development into steps to better understand the gradual development and the different features associated with each level: (i) the biofilm grows at the centre of the drop on the polystyrene; (ii) the biofilm grows following a coffee ring shape; (iii) development of biofilm at the A-L interface (pellicles); (iv) destruction of pellicles; (v) thickening of the biofilm on polystyrene. Observations, emerging from the data, strongly support the idea that the biofilm started to grow at the centre of the drop. This region was characterised by a depletion of oxygen; the cells were stressed and started to grow as biofilms [61]. Then, the biofilm grew at the border of the drop, showing a coffee ring effect, to create a bacterial community able to exploit the higher oxygen availability at the border (oxygen gradient). The growth indicated greater oxygen availability at the border (coffee ring region) which pointed out the gradient along the radial direction of the drop (Figure 1). The coffee ring effect demonstrated the biofilm to thicken at the border. After this stage, the biofilm started to develop at the A-L interface. What is known about the tendency of _P. fluorescens_ to colonise the surface of a (static) liquid is based on the findings of [PERSON] et al. (2016). The authors claimed that the bacteria produced beta-1,4-linked polymers (alginate-based), which have been referred to as pellicles in other works ([PERSON] et al., 2016; [PERSON] et al., 2017). Moreover, the development of biofilms at the A-L interface, compared to the one developed at the polystyrene interface, occurs in a favourable microenvironment where oxygen concentration is high compared to the polystyrene interface ([PERSON] et al., 2016). The outcomes of these experiments demonstrated that at the beginning, the A-L biofilm was composed by a single connected pellicle. Successively, the bacterial community started to destroy it ([PERSON] et al., 2016). The hypothesis is that _P. fluorescens_ started to produce enzymes (like lipase or esterase) that destroyed the EPS in order to use this material for other survival functions. These degrading enzymes stimulated the dispersal of formed pellicles ([PERSON] et al., 2016). When the A-L biofilm starts to be destroyed, it is transformed into several pellicles. These pellicles are reduced into small pieces, resulting in EPS islands floating at the A-L interface (Section 3.4.2). Finally, the destruction of this biomass corresponded to a thickening and reinforcement on the biofilm grown on the polystyrene surface. After a total of 24 h, the biofilm on the polystyrene surface became thick and uniform on the full, delimited area. The coffee ring effect is no longer visible. It is thought that flagella's presence promotes the development of biofilms in batch systems as they are generally associated with motility ([PERSON] et al., 2016; [PERSON] et al., 2017; [PERSON] et al., 2018). Flagella genes and biosurfactant genes have indeed been identified inside the genome sequence of the _P. fluorescens_ ATCC 13525 chosen for this study ([PERSON] et al., 2017). Based on different studies; however, it was found and confirmed that bacteria grow as biofilms on A-L interface, regardless of the absence of flagella, and that cell motility could have a major relevance ([PERSON] et al., 2018).