## 4 Conclusions  
The present study aimed to evaluate the influence of surface materials and nutrient conditions on eGFP production in _E. coli_ biofilms. Three materials were selected and characterized for their topography, morphology, and hydrophobicity. Results revealed that silicone had the highest surface roughness and hydrophobicity compared to PVC and stainless steel. To evaluate the combined effects of these materials and different nutrient conditions on biofilm development and eGFP expression, _E. coli_ biofilms were formed over 9 days and analyzed for cell count, biofilm thickness, biofilm biovolume, eGFP production, and plasmid copy number. Terrific broth was the culture medium that most effectively promoted biofilm growth, as demonstrated by the increased number of biofilm cells, biovolume, and thickness. Although the effect of surface materials on biofilm cell count was not evident, PVC resulted in denser and thicker biofilms after 9 days of incubation. Additionally, the combination of TB culture medium and PVC material led to the highest eGFP expression (particularly on day 1) and plasmid stability. Therefore, the choice of surface materials and nutritional conditions significantly impacts recombinant protein expression in biofilm systems. Since this study evaluates, for the first time, the combined effects of surface properties and nutrient conditions, it represents a significant advancement in the production of recombinant proteins using biofilm systems. These findings highlight the importance of optimizing operational parameters to enhance protein production on a large scale, making them highly relevant at an industrial level. However, the platform selected for biofilm development is a potential limitation of this study. Although microplates represent a high-throughput platform suitable for assays involving a large number of testing conditions, they do not accurately represent real industrial scenarios. In future research, this limitation will be addressed by using continuous systems. Furthermore, the insights gained from this study will be applied to the production of other proteins and value-added compounds using biofilm systems. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app142311029/s1](https://www.mdpi.com/article/10.3390/app142311029/s1)]([https://www.mdpi.com/article/10.3390/app142311029/s1](https://www.mdpi.com/article/10.3390/app142311029/s1)), Figure S1: Representative three-dimensional AFM images of **(a)** SST, **(b)** PVC, and **(c)** SIL; Table S1. Contact angles with water (\(\theta_{W}\)), formamide (\(\theta_{F}\)), and \(\alpha\)-bromonaphthalene (\(\theta_{B}\)); Table S2. Descriptive analysis of the total cells, culturable cells, eGFP production, and plasmid copy number variables; Table S3. Descriptive analysis of the biofilm biovolume, biofilm thickness, and eGFP-expressing biovolume variables. Conceptualization, L.C.G. and F.J.M. ; methodology, A.A. and L.C.G. ; formal analysis, A.A. and R.T.-S.; resources, G.A.M. and F.J.M. ; data curation, A.A. and R.T.-S; writing--original  
Figure 6: Total biovolume **(a)**, average thickness **(b)**, and percentage of eGFP-expressing biovolume **(c)** of _E. coli_ biofilms formed on stainless steel (SST), polyvinyl chloride (PVC), and silicone rubber (SIL) and in Lysogeny broth (LB; ), Terrific broth (TB; ), and M9 ZB broth (M9 ZB; ) media for 9 days. The means \(\pm\) standard deviations (SDs) for three independent experiments are presented. Different lowercase letters indicate statistically significant differences between culture media on each surface (\(p<0.05\)). draft preparation, A.A., F.M.C. and R.T.-S.; writing--review and editing, L.C.G., G.A.M. and F.J.M. ; supervision, L.C.G., G.A.M. and F.J.M. ; funding acquisition, F.J.M. All authors have read and agreed to the published version of the manuscript.