Media and bacterial cell cultivation
All medium components were obtained from Sigma-Aldrich Co. (St Louis, MO, USA) except yeast extract and tryptone which were obtained from BD Diagnostic Systems (Franklin Lakes, NJ, USA). E. coli strains, stored as glycerol stocks at -80°C, were streaked on lysogeny broth (LB; 10 g l-1 tryptone, 5 g l-1 yeast extract, and 5 g l-1 NaCl) agar plates and incubated at 37°C for 14-16 hours.
For shake-flask cultivations, single colonies were picked from LB plates to inoculate 30 ml LB medium in 125 ml conical flasks. The cultures were shaken at 37°C and 280 rpm in a rotary shaker (New Brunswick Scientific, NJ, USA) and used as seed cultures to inoculate 220 ml LB media at 1% (v/v) in 1 L conical flasks. This second seed culture was shaken at 37°C and 280 rpm until a cell density of 0.80 OD600 was reached. Cells were then harvested by centrifugation at 9,000 ×g and 20°C for 10 minutes and resuspended in 30 ml modified M9 production media. The suspended culture was transferred into a 125 ml screwed cap plastic production flasks and incubated at 37°C at 280 rpm in a rotary shaker. Unless otherwise specified, the modified M9 production medium contained 20 g l-1 glycerol, 5 g l-1yeast extract, 10 mM NaHCO3, 1 mM MgCl2,5th dilution of M9 salts mix (33.9 g l-1 Na2HPO4, 15 g l-1 KH2PO4, 5 g l-1 NH4Cl, 2.5 g l-1NaCl ), 1,000th dilution of Trace Metal Mix A5 (2.86 g l-1 H3BO3, 1.81 g l-1 MnCl2•4H2O, 0.222 g l-1 ZnSO4•7H2O, 0.39 g l-1Na2MoO4•2H2O, 79 µg l-1 CuSO4•5H2O, 49.4 µg l-1Co(NO3)2•6H2O), and was supplemented with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). All shake-flask cultivation experiments were performed in triplicate.
For bioreactor cultivations, single colonies were picked from LB plates to inoculate 30 mL super broth (SB) medium (32 g l-1tryptone, 20 g l-1 yeast extract, and 5 g l-1 NaCl) in 125 mL conical flasks. The overnight cultures were shaken at 37°C and 280 rpm in a rotary shaker (New Brunswick Scientific, NJ, USA) and used as seed cultures to inoculate 220 mL SB media at 1% (v/v) in 1 L conical flasks. This second seed culture was shaken at 37°C and 280 rpm for 14-16 hours. Cells were then harvested by centrifugation at 9,000×g and 20°C for 10 minutes and resuspended in 50 mL fresh LB media. The suspended culture was used to inoculate a 1 L stirred tank bioreactor (containing two Rushton radial flow disks as impellers) (CelliGen 115, Eppendorf AG, Hamburg, Germany) at 37°C and 430 rpm. The semi-defined production medium in the batch bioreactor contained 30 g l−1 glycerol, 0.23 g l−1 K2HPO4, 0.51 g l−1 NH4Cl, 49.8 mg l−1 MgCl2, 48.1 mg l−1 K2SO4, 1.52 mg l−1 FeSO4, 0.055 mg l−1 CaCl2, 2.93 g l−1 NaCl, 0.72 g l−1 tricine, 10 g l−1 yeast extract, 10 mM NaHCO3, and 1,000th dilution (i.e., 1 ml l−1) trace elements (2.86 g l−1 H3BO3, 1.81 g l−1 MnCl2• 4H2O, 0.222 g l−1 ZnSO4• 7H2O, 0.39 g l−1 Na2MoO4• 2H2O, 79 μg l−1 CuSO4• 5H2O, 49.4 μg l−1Co(NO3)2• 6H2O) (Neidhardt et al. 1974), and was supplemented with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Microaerobic and semiaerobic conditions were maintained by purging air into the headspace and bulk culture, respectively, at 0.1 vvm, designated as aeration level I (AL-I) and II (AL-II). Aerobic conditions were maintained by purging air into the bulk culture at 1 vvm (AL-III). The pH of the production culture was maintained at 7.0 ± 0.1 with 30% (v/v) NH4OH and 15% (v/v) H3PO4.