## 1 Introduction  
_Escherichia coli_ has been one of the most used organisms for heterologous protein production due to its inexpensive and fast high-cell density cultivation, well-known genetics, and large number of cloning vectors [1, 2, 3]. In addition to the host strain, factors such as the expression vector, cultivation conditions (medium composition, temperature, pH, and induction conditions), and purification strategies are essential for the successful production of heterologous proteins [4]. _E. coli_ plasmids have been the preferred expression vectors because of the high gene dosage that can be attained and the capability to control the levels of gene expression through inducible promoters [5]. Although a high plasmid copy number (PCN) may ensure a high gene dosage, this does not always mean a high level of target heterologous protein [5, 6]. Studies in suspension cell cultures have demonstrated that high plasmid copy number imposes a metabolic burden on the host cell, which reduces the bacterial growth rate and favors plasmid instability, thereby decreasing the overall protein yield [7, 8]. The use of biofilm cells in recombinant protein production has been scarcely studied, but some promising results have been obtained by our research group in the last few years [6; 9; 10; 11]. The biological organization of biofilms provides them with many advantages over cells grown in suspension cell cultures, including high cell density, protection against hostile conditions, and operation stability, particularly when biofilms are used in biotechnological processes for the production of value-added compounds [12]. In addition, the presence of plasmids has been shown to stimulate biofilm formation and lead to a higher production of recombinant proteins compared to planktonic cells [10; 11; 12; 13; 14; 15]. Our group has demonstrated that cells expressing eGFP (enhanced green fluorescent protein) formed more biofilm than non-expressing cells [10]. Moreover, _E. coli_ biofilms were able to produce eGFP at a much higher level (30-fold) than their planktonic counterparts [10]. Furthermore, it has been shown that biofilm cultures have a beneficial effect on the maintenance of high and medium copy number plasmids [6; 16]. [PERSON] et al. [16] reported that continuous biofilm cultures of _E. coli_ ATCC 33456 containing the pEGFP plasmid (a pUC family vector) maintained a high PCN over extended periods, while in chemostat a significant plasmid loss was observed. More recently, we showed that although the PCN was higher in planktonic cells, a significant PCN reduction (around 89%) was observed in planktonic cells after 5 days of flow cell operation, while no plasmid loss was observed in _E. coli_ JM109(DE3) biofilms [6]. Different strategies have been explored to achieve maximum yields of heterologous proteins in suspended cell reactors, including by (1) changing the expression vector and/or the host, (2) optimizing the culture parameters (medium, pH, and temperature) and induction conditions (inducer concentration and temperature) of the recombinant strain, (3) co-expressing other genes such as chaperones, and (4) adjusting the synthesis of stress-related sigma factors and/or deleting specific gene sequences such as proteases, which may promote both the quantity and quality of the desired protein [2; 3; 5; 17; 18; 19]. Nonetheless, the investigation of productive biofilms remains scarce. One of the parameters that can affect both biofilm formation and the level of heterologous protein expression is the culture medium composition. Several reports have demonstrated that the nutritional medium content affects biofilm formation [11; 20; 21; 22]. Some authors have shown that an increase in nutrient levels promotes biofilm formation [12; 13]. However, contradictory results showing that high nutrient concentration inhibits biofilm formation were also reported [14; 15]. Regarding the effect of nutrient medium composition on heterologous protein production, most of the studies were performed in planktonic conditions [23; 24; 25].