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.

Real‐time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control

“…Liquid and gas Used RAMOS to measure in the headspace and rotating flexi-tube optical sensor to monitor dissolved O 2 , CO 2 , and culture pH Hansen, Jacob, Luchterhand, and Büchs (2012) Fluorescence-based method for monitoring CO 2 and O 2 in the culture broth and in the headspace of an Erlenmeyer flask and broth pH as well Ge and Rao (2012) Circulation direct monitoring and sampling system-a system that continuously extracts and measures liquid (by electrode) and gas-phase samples Takahashi et al (2017) Takahashi and Aoyagi (2018a, 2018b, and 2018c Abbreviations: dCO 2 , dissolved carbon dioxide; OTR, oxygen transfer rate; RAMOS, respiration activity monitoring system.…”

“…The fluorescencebased method has been reported to measure O 2 and CO 2 in the headspace as well as in culture broth. The effect of fluorescent pigment has always been debated (Ge et al, 2005;Gupta & Rao, 2003 (Takahashi & Aoyagi, 2018a, 2018bTakahashi, Sawada, & Aoyagi, 2017). However, it is critical to prevent cells from clumping to avoid the clogging of the liquid circulation part.…”

“…DO was monitored by an optical sensor that is provided by Scientific Industries, Inc. The detailed description of the DO optical sensor can be found in the literature (Tolosa, Kostov, Harms, & Rao, 2002). The cultures were sampled once a day for optical density measurement.…”

“…The effect of fluorescent pigment has always been debated (Ge et al, ; Gupta & Rao, ). Takahashi et al developed a circulation‐based direct monitoring and sampling system with a circulation bypass component in the measuring site tilted in such a way that cells do not accumulate and the air bubbles are well dispersed (Takahashi & Aoyagi, , ; Takahashi, Sawada, & Aoyagi, ). However, it is critical to prevent cells from clumping to avoid the clogging of the liquid circulation part.…”

Erratum to: 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

“…Following publication of the original article (Takahashi et al 2017 ), the authors reported that there was a mistake in the legend of Figure 2, which in the published article reads as follows:…”