Development of a circulation direct sampling and monitoring system for O2 and CO2 concentrations in the gas-liquid phases of shake-flask systems during microbial cell culture

Abstract

Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO₂ and O₂ in the gas-liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of Escherichia coli using this set-up indicated that a high concentration of CO₂ accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5-9.0 h). By packing a CO₂ absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO₂ from the headspace. It was posited that removing CO₂ from the headspace would suppress increases in the dissolved CO₂ concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5-12.0 h) was extended, the U.O.D.₅₈₀ and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of E. coli cells without changing the composition of the medium, temperature, and shaking conditions.

Keywords: CO2; Escherichia coli; Headspace; Real-time monitoring; Sampling; Shake-flask culture.

Fig. 1

Schematic flow diagram of the circulation direct monitoring and sampling system for shake-flask (CDMSS). A Culture plug penetrated with a needle; B Erlenmeyer flask with branches on the bottom; C-a the gas circulation system (normal); C-b the gas circulation system (with CO₂ absorbent); D the liquid circulation system; 1 connector; 2 0.22 µm filter; 3 flow meter; 4 diaphragm-type pump; 5 liquid measurement unit; 6 gas sampling unit; 7 check valve; 8 column packed with CO₂ absorbent; 9 peristaltic pump; 10 liquid measuring unit; 11 liquid sampling unit

Fig. 2

Real-time CO₂ and O₂ concentrations in normal and specialised shake-flask culture of E. coli IFO3301. a In the headspace; b in the culture medium. Symbols: circles oxygen; triangles carbon dioxide; closed normal shake-flask culture; open specialised shake-flask culture (with removal of CO₂ in the headspace). Shake-flask culture conditions were 100 mL of LB medium at 30 °C, 200 rpm shaking frequency and 70 mm shaking diameter, 500 mL Erlenmeyer flask capped with a culture plug and equipped with CDMSS

Fig. 3

Changes in culture factors in the two shake-flask cultures. a Cell biomass (U.O.D.₅₈₀); b pH and acetic acid concentration. Symbols: closed circles control (normal shake-flask culture); open circles CO₂ absorbent-equipped (specialised shake-flask culture). Symbols in b circles pH; triangles acetic acid concentration