## 4 Discussion  
Heterologous protein production using _E. coli_ biofilms has shown to be advantageous for many reasons, including higher plasmid copy number [16] and a higher level of protein production compared to their planktonic counterparts [10,39]. However, the most important goal of heterologous protein production is to achieve high protein production at low cost. Several parameters, such as nutrient medium composition, temperature, hydrodynamic conditions, and surface properties can affect biofilm formation [11,22,40] and consequently influence the final protein yield. In a previous study [11], our research group compared the effect of two culture media--a diluted medium (DM) and LB--and concluded that biofilms grown in LB produced more eGFP than those grown in DM. In this preliminary work from 2017, primacy was given to culture media typically used to promote biofilm growth such as DM [22,33], while here, there was a concern in testing media recommended for the high expression of recombinant proteins in _E. coli_ such as the M9 ZB. Planktonic results showed a higher number of culturable cells in M9 ZB compared to LB medium. This could be related to the presence of glucose in M9 ZB medium, which is a compound that is absent in the LB medium used. Previously, [PERSON] et al. [41] also found that a culture of _E. coli_ DH5\(\alpha\) cells harboring the recombinant plasmid pMAL-c2X achieved higher cell densities in media with a high concentration of glucose, such as Tryptone Yeast (TY) medium, Terrific Broth (TB), and Super Broth (SB). Biofilm formation was also favored in the M9 ZB medium, as indicated by the higher values of both culturable and total cells when compared to LB. Several studies have shown that the nutritional content of the medium, namely carbon and energy substrates, affects biofilm formation [42,43]. [PERSON] and [PERSON] [44] observed that using a minimal salts medium (MSM) supplemented with glucose resulted in a stable biofilm with shorter bacterial cells and a thicker extracellular matrix. [PERSON] et al. [13] have also reported that increasing the glucose concentration of culture medium can promote the growth of _E. coli_ nutrient-depleted biofilms. Concerning heterologous protein production, a higher number of cells expressing eGFP and higher specific eGFP concentrations were found in LB for both planktonic and sessile cells. Complex media, such as LB, contain yeast extract and tryptone that are known to provide biosynthetic precursors, such as vitamins and amino acids, that support protein expression in _E. coli_[26,27]. Furthermore, yeast extract can also be responsible for reducing the cellular stress (protease production) during the synthesis of recombinant proteins [45]. Similar to what was observed in the present work with LB, [PERSON] et al. [26] found that a synthetic medium containing yeast extract and tryptone resulted in higher Streptokinase protein concentration in _E. coli_, without increasing cell density [26]. The lower eGFP expression in M9 ZB medium could be related to the presence of glucose in the medium by repressing the lac promoter [30,46]. In the present work, a pET system was used, where the eGFP gene is transcribed by T7 RNA polymerase, which is regulated by the lacUV5 promoter [4]. It has been shown that glucose reduced the basal expression by reducing cyclic adenosine monophosphate (cAMP) levels, resulting in a low transcription from lacUV5 promoter [46]. [PERSON] et al. [47] confirmed that glucose supplementation repressed the expression of recombinant human granulocyte colony-stimulating factor (hG-CSF). On the other hand, it was revealed that the addition of yeast extract to the culture media increased the background expression from the lac promoter [48,49]. Regardless of the culture medium used, there was an increase in the percentage of eGFP-expressing cells in both planktonic and biofilm between days 5 and 7. This phenomenon may be associated with the higher number of culturable cells also detected in the same period.