Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

Abstract Background Screening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli. Results In combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation. Conclusions The newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations

MOESM1 of Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

Additional file 1. Set-up of the membrane-based fed-batch shake flask. (a) The offline and (b) The online membrane-based fed-batch shake flask

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Additional file 3. Comparison of calculated and measured values at varied initial reservoir glucose concentrations with the membrane-based fed-batch shake flask. Depiction of (a) + (b) Measured OTR; (c) + (d) Measured and calculated amount of metabolized glucose; (e) + (f) Measured and calculated amount of cell dry weight; (g) + (h) Measured and calculated amount of metabolized ammonium and accumulated glucose. For calculations the stoichiometric equation C6H12O6 + 0.57 NH3 + 2.34 O2 → 3.38 CH1.7O0.43N0.17 + 2.62 CO2 + 3.99 H2O was used. Calculations until the abrupt OTR increases are based on measured total oxygen consumed values, calculations after the abrupt OTR increase are based on the calculated glucose release; Cultivation conditions: Wilms-MOPS-mineral medium (culture medium: 200 mM MOPS with 7 g/L initial ammonium sulfate, reservoir: 250/375/500/750 g/L glucose, blue dextran concentration: 1 g/L), temperature: 37 °C, shaking frequency: 350 rpm, shaking diameter: 50 mm, initial culture medium volume: 10 mL, inoculation OD600: 0.5, reservoir filling volume: 2 mL, dialysis membrane: Reichelt 10-20 kDa, membrane area: 18.1 mm2; Strain: E. coli BL21 (DE3) pRhotHi-2-EcFbFP; Concentration values have been volume-corrected

MOESM2 of Systematic evaluation of characteristics of the membrane-based fed-batch shake flask

Additional file 2. Effect of membrane thickness of a dialysis membrane on oxygen transfer rate in E. coli fed-batch cultivations (duplicates). Variation of membrane thickness between 28 µm (Reichelt (2) dialysis membrane) and 42 µm (Reichelt (1) dialysis membrane); cultivation conditions: Wilms-MOPS-mineral medium (0.2 M MOPS, 250 g/L glucose in the reservoir), temperature: 37 °C, shaking frequency: 350 rpm, shaking diameter: 50 mm, culture broth volume: 10 mL, inoculation OD600: 0.5, reservoir filling volume: 2 mL, membrane area: 18.1 mm2; strain: E. coli BL21 (DE3) pRhotHi-2-EcFbFP

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Additional file 7. Uncorrected and corrected cell dry weight results for multicomponent fed-batch cultivations. (a) Direct cell dry weight measurements of culture medium; (b) Cell dry weight measurements corrected for evaporation and water back-diffusion; Cultivation conditions: Wilms-MOPS-mineral medium (culture medium: 200 mM MOPS with 7 g/L initial ammonium sulfate, reservoir: 250/375/500/750 g/L glucose, 40 g/L ammonium carbonate, blue dextran concentration: 1 g/L), temperature: 37 °C, shaking frequency: 350 rpm, shaking diameter: 50 mm, initial culture medium volume: 10 mL, inoculation OD600: 0.5, reservoir filling volume: 2 mL, dialysis membrane: Reichelt 10-20 kDa, membrane area: 18.1 mm2; Strain: E. coli BL21 (DE3) pRhotHi-2-EcFbFP

MOESM8 of Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

Additional file 8. Effect of dipotassium phosphate concentration variation on OTR in cultivations with additional glucose and ammonium carbonate in the reservoir. Cultivation conditions: Wilms-MOPS-mineral medium (culture medium: 200 mM MOPS with 7 g/L initial ammonium sulfate, reservoir: 500 g/L glucose, 40 g/L ammonium carbonate, varied dipotassium phosphate concentration, blue dextran concentration: 1 g/L), temperature: 37 °C, shaking frequency: 350 rpm, shaking diameter: 50 mm, initial culture medium volume: 10 mL, inoculation OD600: 0.5, reservoir filling volume: 2 mL, dialysis membrane: Reichelt 10-20 kDa, membrane area: 18.1 mm2; Strain: E. coli BL21 (DE3) pRhotHi-2-EcFbFP

MOESM9 of Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

Additional file 9. Average values and standard deviations for validation of reproducibility. Depiction of (a) Eight replicates within one experiment; (b) Five replicates from independent experiments; Cultivation conditions: Wilms-MOPS-mineral medium (culture medium: 200 mM MOPS with 7 g/L initial ammonium sulfate, initial reservoir glucose concentrations: 250 g/L, blue dextran concentration: 4 g/L, temperature: 37 °C, shaking frequency: 350 rpm, shaking diameter: 50 mm, initial culture medium volume: 10 mL, inoculation OD600: 0.5, reservoir filling volume: 2 mL, dialysis membrane: Reichelt 10-20 kDa, membrane area: 18.1 mm2; Strain: E. coli BL21 (DE3) pRhotHi-2-EcFbFP