3.4. The role of the Oxypump stoppers over E. coli BL21-Gold (DE3) rSMD cultures grown in flasks shaken by the RAMbio
The role of the active gas transfer generated by the Oxypump stoppers was further analyzed. For this, we used a modified Oxypump stopper, where the pumping movement of the bellows element was cancelled (Fig. 2B). By using this special closure, the kLa values were not affected (Fig. S4A and B).
Except for the closures used, E. coli was grown under the same conditions as the previous cultures (Section 2.7– 2.8). It was incubated in the RAMbio system at 10 g, having a kLa value of 96.1 ± 3.4 h−1 ( Table 5). The results are in Fig. 6E–H showing BI6727 growth, glucose uptake, DOT profiles and pH measurements in comparison to the cultures evaluated at the same operational conditions and the normal Oxypump stoppers. Main parameters are summarized in Table 5.
Even when the E. coli cultures where uninduced, the stress associated with producing the recombinant rSMD and maintaining the plasmid led to less biomass being produced than with the wild type . E. coli BL21 DE3 gold (wild type) was grown with the same conditions as previous experiments ( Fig. 6E–H), in absence of ampicillin. Table 5 summarizes the main parameters of those cultures, while the biomass growth, glucose uptake and DOT profiles can be analyzed from Fig. S5A–C. The performance of those cultures is clearly superior, as they reached higher specific growth rate and specific glucose uptake. In addition, they were enhanced when growing in the RAMbio system, due to the increase oxygen supply. The higher final pH values indicate that the acetate production may be diminished or even avoided, therefore using more glucose to biomass production.