Biolite 25 cm2 plug sealed tissue culture flasks

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental cultures was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentrations: chloramphenicol, 10 μg mL⁻¹; spectinomycin, 25 μg mL⁻¹; erythromycin, 25 μg mL⁻¹; and kanamycin, 25 μg mL⁻¹). All experimental cultures were prepared in quadruplicates. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliters of culture were sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The pH of experimental cultures was measured with MColorpHast™ pH-indicator strips (pH 6.5–10) (Merck) and the cultures pH were adjusted to the range between 7–8 using 37% HCl (Sigma-Aldrich).

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental culture was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentration: chloramphenicol 20 μg mL⁻¹, spectinomycin 50 μg mL⁻¹, and kanamycin 50 μg mL⁻¹). All experimental cultures were prepared in triplicate. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliter of culture was sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The cultivation was terminated when the isobutanol production in the culture started to decrease.