## 1 Introduction  
Over the past 50 years, extensive research on the production of engineered proteins using various expression systems has revolutionized the biotechnology field by enabling the expression and purification of desired proteins and peptides in large amounts for an assortment of industrial and clinical applications [1; 2; 3]. This process typically involves cloning the gene encoding the target protein into an expression system, followed by its transformation into a host [4; 5]. Designing an efficient recombinant protein (RP) production process requires careful consideration of several key factors to ensure the highest levels of protein synthesis [6], including the selection of the expression system (generally plasmid vectors) and the characteristics of its genetic elements (e.g., promoter, origin of replication, affinity tags, terminator, and selection marker), and the choice of host cells (e.g., bacteria, filamentous fungi, yeasts, mammalian cells, plants, and insect cells) [7; 8; 9; 10]. _Escherichia coli_ remains the preferable host for heterologous protein production due to its well-recognized and advantageous characteristics over other expression hosts [11; 12; 13; 9; 14]. To date, most RP production has been performed using bacterial cultures in suspension. However, this imposes a metabolic burden on cells, as it diverts precursors and energy towards the maintenance and expression of foreign genes [5; 15; 16]. In planktonic cells, this metabolic burden may inhibit cellular growth, promote plasmid instability, and induce metabolic changes and stress responses in the host cells, ultimately decreasing overall protein yield and activity [17; 18; 19; 20]. During the past decades, microbial biofilms have been proposed as a reliable alternative to surpass some limitations found in suspended cell cultures, offering higher biomass density and operational stability [15], and demonstrating great potential for the production of a wide range of bulk and fine chemicals [21; 22; 23]. Our research group has been studying the expression of the heterologous enhanced Green Fluorescent Protein (eGFP) from the pFM23 plasmid in _E. coli_ JM109(DE3) biofilms, as well as the effects of various operational parameters on eGFP productivity and biofilm formation [17; 24; 25; 26; 27; 28]. Some parameters such as hydrodynamics, surface properties, nutritional conditions, and chemical induction have been explored since they can affect biofilm development and, consequently, RP expression [29]. In fact, surface properties (e.g., charge, chemistry, topography, hydrophobicity, and roughness) are important in bacterial adhesion and subsequent biofilm development [30; 31; 32; 33; 34]. Surfaces used in biofilm reactors should promote microorganism adhesion to increase biomass density. Additionally, they should be inexpensive, widely available, and not require harsh conditions for biofilm detachment to facilitate downstream processes [35; 36]. Polymers are frequently the material of choice due to their moderate costs and the ease with which their composition can be tailored for specific applications [37]. The nutritional composition of the culture medium also plays a crucial role in the expression, solubility, and secretion of RPs [6; 27; 38; 39; 40; 41], and must be carefully selected to meet the cellular and process requirements [4]. The medium typically includes carbon sources, such as glucose or glycerol, and nitrogen sources, such as yeast extract, tryptone, and peptone [42], and can also be supplemented with amino acids and other precursors (e.g., cofactors and vitamins) to prevent shortages that typically reduce cell growth rate and RP production [4]. Glucose is the preferred carbon source for _E. coli_ during the initial growth period [43], as it prevents the uptake and consumption of lactose, and consequently, the auto-induction of _lac_-controlled protein expression [9; 42; 43; 44].