Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

Abstract

BackgroundThere are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na2CO3. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.ResultsThe controlled-release discs presented here were successfully used to cultivate E. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all.ConclusionsThese novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes.

Gracilimonas rosea sp. nov., isolated from tropical seawater, and emended description of the genus Gracilimonas

“…The pH range for growth was also determined by assessing changes in the OD 600 in MB adjusted to pH 5.0-10.0 (in increments of 0.5 pH units), incubated at 35 u C for 7 days. The following buffers were used for pH experiments: citrate/phosphate (pH 5.0 and 5.5), MOPS (Sigma) (pH 6.0-8.0), boric acid/borax (pH 8.5 and 9.0), carbonate/bicarbonate (pH 9.5 and 10.0), borax/NaOH (pH 9.5 and 10.0) and Tris/HCl (pH 8.5-10.0), each at a final concentration of 50 mM (Scheidle et al, 2011;Jang et al, 2013). The pH of a subsample of autoclave-sterilized medium was measured before inoculation of cells to check for changes in pH.…”

Rhizobium populi sp. nov., an endophytic bacterium isolated from Populus euphratica

“…A small amount of the purified colony was inoculated in 50 ml King A liquid medium for 3 days on a rotary shaker at 150 r.p.m., 28 u C, to determine the optimum temperature for growth by measuring OD 600 using a spectrometric method (Optizen 2120UV; Mechasis). The pH range (pH 5.0-10.0, intervals of 0.5 pH units) for growth was determined by assessing changes in OD 600 over the incubation period (up to 7 days) in King A broth at 28 u C. Prior to autoclaving, the pH of the medium was adjusted using appropriate biological buffers: citrate/phosphate (pH 5.0 and 5.5), MOPS (Sigma) (pH 6.0-8.0), boric acid/borax (pH 8.5 and 9.0), borax/ NaOH (pH 9.5 and 10.0) and Tris/HCl (pH 8.5-10.0), each at a final concentration of 50 mM (Scheidle et al, 2011). After autoclaving and cooling, the pH of subsamples of each medium was measured, before inoculation with cells.…”

Characterization of antibacterial polyethersulfone membranes using the respiration activity monitoring system (RAMOS)

“…It was used for different applications in process development, media optimization and screening of microorganisms (Stö ckmann et al, 2003;Jeude et al, 2006;Klement et al, 2011). Several publications characterized the growth of the model microorganism E. coli in RAMOS shake flask cultures (Losen et al, 2004;Scheidle et al, 2007;Huber et al, 2011;Scheidle et al, 2011).…”