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.