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Biofilm research is usually focused on the prevention or control of biofilm formation. Recently, the significance of the biofilm mode of growth in biotechnological applications received increased attention. Since biofilm reactors show many advantages over suspended cell reactors, especially in their higher biomass density and operational stability, bacterial biofilms have emerged as an interesting approach for the expression of specific proteins. Despite the potential of biofilm systems, recombinant protein production using biofilms has been scarcely investigated for the past 25 years. Our group has demonstrated that E. coli biofilms were able to produce a model recombinant protein, the enhanced green fluorescent protein (eGFP), at much higher levels than their planktonic counterparts. Even without optimization of cultivation conditions, an attractive productivity was obtained, indicating that biofilm cultures can be used as an alternative form of high cell density cultivation (HCDC). E. coli remains one of the favorite hosts for recombinant protein production and it has been successfully used in metabolic engineering for the synthesis of high value products. This review presents the advantages and concerns of using biofilms for the production of recombinant proteins and summarizes the different biofilm systems which have been described for this purpose. The relative advantages and disadvantages of the four microbial hosts tested for recombinant protein production in biofilms (two bacteria and two filamentous fungi) are also discussed.

In the present study, the effects of different nutrient media on the development of Escherichia coli biofilms and the production of a heterologous protein were examined. E. coli JM109(DE3) cells transformed with pFM23 plasmid carrying the gene for enhanced green fluorescent protein (eGFP) expression were used. Cells were grown in two different culture media, Lysogenic Broth (LB) and M9ZB, in a flow cell system for 10 days. Epifluorescence microscopy, fluorimetry, and a high-performance liquid chromatography (HPLC) method based on hydrophobic interaction chromatography (HIC) were used to assess bacterial growth, plasmid copy number (PCN), and eGFP production in both planktonic and biofilm cells. The results showed that biofilm development was favored in M9ZB medium when compared with LB. However, the number of eGFP-expressing cells was higher in LB for both planktonic and sessile states (two-fold and seven-fold, respectively). In addition, the PCN in biofilm cells was slightly higher when using LB medium (on average, 29 plasmids per cell versus 20 plasmids per cell in M9ZB), and higher plasmid stability was observed in biofilms formed in LB compared to their planktonic counterparts. Hence, E. coli biofilms grown in LB enhanced both plasmid stability and capacity to produce the model heterologous protein when compared to M9ZB.

Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a model recombinant protein (enhanced green fluorescent protein, eGFP) between planktonic and biofilm cells. The fluorometric analysis revealed that when the system was in steady state, the average specific eGFP production from Escherichia coli biofilm cells was 10-fold higher than in planktonic cells. Additionally, epifluorescence microscopy was used to determine the percentage of eGFP-expressing cells in both planktonic and biofilm populations. In steady state, the percentage of planktonic-expressing cells oscillated around 5%, whereas for biofilms eGFP-expressing cells represented on average 21% of the total cell population. Therefore, the combination of fluorometric and microscopy data allowed us to conclude that E. coli biofilm cells can have a higher recombinant protein production capacity when compared to their planktonic counterparts.