Biostat b plus fermenter

In the symbiotic system, inoculum volume of C. acetobutylicum TSH1 was 5 %, and B. cereus TSH2 was 0.5 % if not otherwise indicated. All of the inoculum size used volume ratio (vol %). To keep constant inoculation volume, the seed cultures were diluted to similar OD value before inoculation. Shake flask culture was carried out in 100 mL shaken flask with a rubber stopper, and working volume was 60 mL. The fermentation was performed statically at 37 °C without anaerobic treatment. Bioreactor fermentation was carried out in 5 L Biostat B plus fermenter (Sartorius company, Germany) containing 3 L P₂ medium (55–60 g/L initial glucose) at 37 °C. Anaerobic fermentation was performed with N₂ flushing pretreatment. Microaerobic fermentation was achieved by static culture or flushing with air at a rate of 0.15 L/min (0.05 vvm). Samples were taken at regular intervals to analyze biomass, substrate and solvents concentration.

All signal-peptide-free (but otherwise full-length native) encoding sequences of ChryC1–3 were cloned into pET28a(+) from Novagen (Madison, WI, USA) by restriction-free cloning [15 (link)]. The chosen vector provided an N-terminal His6-tag to each encoding sequence. All proteins were recombinantly produced in Escherichia coli BL21-Gold (DE3) pLysS (Novagen) cultured at 30 °C in a 5 L BIOSTAT B plus fermenter (Sartorius, Goettingen, Germany) using lysogeny broth (LB) Lennox medium additionally containing 24 mM (NH₄)₂HPO₄, 35 mg·L⁻¹ kanamycin and 0.001% (v/v) Breox FMT 30 antifoam (Cognis, Monheim, Germany). Expression was induced by adding 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at an optical density (600 nm) between 3.0 and 3.5. Cells were harvested after expression for 4 h at 30 °C and suspended in buffer (50 mM TRIS/HCl, 100 mM NaCl, pH 8). Recombinant protein production was confirmed by comparing protein composition in E. coli cells before and after gene expression induction by SDS-PAGE (data not shown).

Bacterial cultures were performed in duplicate in 2 L Biostat B plus fermenter (Sartorius, Melsungen, Germany) filled with glucose (20 g L^-1) containing-CDM or the same medium supplemented with 5 g L^-1 (34 mM) glutamate and/or 5 g L^-1 (29 mM) arginine and/or 20 g L^-1 (149 mM) malate. Cultures were incubated at 30°C in anaerobiosis, obtained by slight N₂ overpressure. pH was maintained at 6.6 by KOH addition until cultures reached OD₅₈₀ = 1, in order to reach enough biomass for further analytical procedures, and then pH was not regulated anymore. Bacterial growth was monitored by measurement of OD₅₈₀ (Libra S11, Biochom, 1 Unit of absorbance is equivalent to 0.3 g L^-1). Samples were collected every 30 min for HPLC determination of metabolite concentration in the growth medium.