## 3 Results and Discussion
### Effect of Surface Material and Nutrient Composition on Biofilm Formation and eGFP Expression
#### 3.2.4 Biofilm Architecture  
The influence of surface material and nutritional conditions on _E. coli_ biofilm development and eGFP expression was also assessed using confocal microscopy. Figure 5 illustrates representative three-dimensional (3D) structures of _E. coli_ biofilms formed on SST, PVC, and SIL in LB, TB, and M9 ZB media, after 9 days of incubation. _E. coli_ formed denser and thicker biofilms on PVC material (Figure 5, middle column), followed by SIL and SST surface (right and left columns, respectively), as demonstrated by the lateral shadow projections. Concerning the culture media, TB resulted in denser and thicker biofilms (Figure 5, middle row) compared to those grown on LB or M9 ZB media (top and bottom rows, respectively). Additionally, in the biofilms with greater thickness (Figure 5e,h), a thin layer of non-eGFP-expressing cells (red cells) was observed at the bottom, covered by a thicker layer of eGFP-expressing cells, as shown in the sectional views below the 3D projections. The presence of non-eGFP-expressing cells at the bottom of the biofilm may be attributed to their limited access to nutrients and oxygen [28; 74]. In biofilms with lower thickness (Figure 4a,g), some dispersed non-eGFP-expressing cells were also observed.  
This qualitative analysis was confirmed through the biofilm biovolume, thickness, and eGFP-expressing biovolume parameters estimated from confocal image analysis (Figure 6). Results demonstrated that biofilms formed on PVC were more developed, with up to 2-fold higher biovolume compared to those formed on SST (Figure 6a). In turn, biofilms grown on SIL exhibited a slightly lower biovolume compared to PVC (up to 1.4-fold lower biovolume). Concerning culture media, TB was the medium that most promoted biofilm growth, resulting in a higher biovolume compared to LB or M9 ZB media (\(p<0.05\)). These differences were particularly noticeable for biofilms formed on PVC and SIL materials. Additionally, biofilms formed on PVC displayed, on average, 2.43- and 1.78-fold greater thickness compared to those on SST and SIL surfaces, respectively (Figure 6b). Furthermore, TB was the medium in which biofilms showed the highest thickness (up to 400 \(\upmu\)m; \(p<0.05\)). For the percentage of eGFP-expressing biovolume (Figure 6c), this was greater for biofilms formed on PVC (on average 69%), followed by SIL and SST (on average 61% and 56%, respectively). TB was the medium where higher eGFP expression values were attained, achieving on average 72% of biovolume expression.  
Figure 5: _E. coli_ biofilms formed on stainless steel (SST; (**a,d,g**)), polyvinyl chloride (PVC; (**b,d,h**)), and silicone rubber (SIL; (**c,f,i**)) and Lysogeny broth (LB; (**a–c**)), Terrific broth (TB; (**d–f**)), and M9 ZB broth (M9 ZB; (**g–i**)) media for 9 days. These representative images were obtained from confocal _z_-stacks using the IMARIS 9.3. software and present an aerial, three-dimensional (3D) view of the biofilms. The black shadow on the right represents the vertical projection of biofilm, while the sectional view below the 3D projection represents the 2D spatial distribution of cells expressing eGFP (green cells; green arrow) and non-expressing cells (red cells; red arrow) along the vertical (z) biofilm position. The white scale bars represent 50 \(\upmu\)m.