## 3 Results  
In this study, two different nutrient media were used with the aim of comparing eGFP production and plasmid stability in biofilm and planktonic populations growing in a flow cell system. The planktonic fraction was monitored by quantifying the total and culturable cells (Figure 2A,C, respectively). Analysis of the planktonic total cells (Figure 2A) indicated that there were very few statistical differences in _E. coli_ growth in both media (only one of the eight experimental points has \(p\)\(<\) 0.05). Despite the few statistical differences, from day 5 onwards, it seems to be a higher cell number of bacteria when growing in LB medium, with on average 29% more cells being present in LB than in M9 ZB until the end of the experiment. Regarding the number of planktonic culturable cells (Figure 2C), similar behavior was observed for both media, but slightly higher CFU mL\({}^{-1}\) values were obtained in M9 ZB in all experimental points (on average 62%) when compared to LB. The impact of nutrient medium composition on biofilm development was assessed by quantifying total and culturable cells and determining the biofilm thickness (Figure 2B,D,E, respectively). Globally, the amount of biofilm total cells (Figure 2B) was higher for M9 ZB (on average 8.25 log cells cm\({}^{-2}\)) than for LB (on average 8.0 log cells cm\({}^{-2}\)) with statistically significant differences for five of the eight experimental points (\(p\)\(<\) 0.1). When comparing biofilm culturable cells (Figure 2D), higher values were also obtained for M9 ZB (on average 65%) with statistical differences in most time points (\(p\)\(<\) 0.1 for days 6 and 10, and \(p\)\(<\) 0.05 for days 3, 5, 7 and 9). Biofilm thickness (Figure 2E) was similar between growth media until day 5, but thereafter, the biofilms developed in M9 ZB were thicker than biofilms formed in LB (176 \(\upmu\)m versus 161 \(\upmu\)m, respectively), which is most likely a result of their higher cell density (Figure 2B,D). Looking at the results of heterologous protein expression (Figure 3), the number of planktonic cells expressing eGFP relative to the number of total cells (Figure 3A) was slightly higher for LB medium in most experimental points. The number of eGFP-expressing cells in both media increased until day 6 when on average 27% of the total cells produced eGFP. Regarding the percentage of eGFP-expressing cells in biofilm (Figure 3B), it can be seen that in M9 ZB, this parameter remained almost constant throughout the experiment (around 7%, with the maximum value of 13% on day 6), while in LB, a strong increase was observed until day 6 when 66% of the total cells expressed eGFP (\(p\)\(<\) 0.05). From day 6 onwards, the amount of expressing cells decreased about 20% and was nearly similar until the end of the experiment. Furthermore, in the LB medium, the number of sessile cells expressing eGFP was about 54% higher than in _E. coli_ suspensions, while in M9 ZB, the number of expressing cells in the biofilm was similar to its planktonic counterparts (Figure 3A,B). The specific eGFP values (Figure 3C,D) showed that for both planktonic and biofilm states, the cells grown in LB synthesized more protein than cells grown in M9 ZB. On average, the eGFP production in LB medium was eight-fold and three-fold higher for planktonic and biofilm cells, respectively, than in M9 ZB. Additionally, in M9 ZB, the eGFP levels remained practically constant over time for both planktonic and biofilm states (around 0.31 fg cell\({}^{-1}\) and 1.51 fg cell\({}^{-1}\), respectively), whereas in LB, the specific eGFP concentration increased until days 5 and 7 for planktonic and biofilm environments, respectively, reaching a maximum of 4.31 fg cell\({}^{-1}\) and 15.96 fg cell\({}^{-1}\), and then it started to decrease, reaching the steady state.