2.6 Succinic acid production from the HPAC-pretreated pine
Mineral salts (0.5 g L-1KH2PO4, 0.5 g L-1K2HPO4, 0.5 g L-1MgSO4·7H2O, 6.0 mg L-1FeSO4·7H2O, 4.2 mg L-1MnSO4·H2O, 0.2 mg L-1biotin, 0.2 mg L-1 thiamine) were added to the
hydrolysates and adjusted to pH 7.5 with 1.0 N
NaOH.[5] The ΔldhA -6 mutant of C.
glutamicum was pre-cultured in 20 mL of LB medium at 30 °C overnight.
The cells were subsequently transferred to 0.5−1.5 L LB medium and
cultured at 30 °C for 24–48 h (approximately 2.5–3.0 g
L-1, CDW). After harvest, the cells (5.36–26.89 g
L-1) were added to the 100 mL hydrolysates and 400 mM
sodium bicarbonate was added to retain semi-anaerobic condition. The
fermentation to produce succinic acid was performed at 200 rpm and 30 °C
in a shaking incubator. The pH of the reaction solution was checked and
maintained at 7.2–7.5 by adding sodium hydroxide after each sampling.
A fed-batch process was initiated with 4% hydrolysate (initial
hydrolysate) and approximately 30 g L-1 CDW of
[Psod:sucE- ΔldhA ] transformant. After 24 h of fermentation,
20 mL of the 20% hydrolysate as a feed was added. The fermentation was
conducted using the same method described above. The yield of succinic
acid was calculated based on the followed equation:
Yield of succinic acid (%) = St / (Gi +
Gf) × 100
Where St is total amount of succinic acid produced in
fed-batch system, and Gi and Gfrepresent the glucose concentration of the initial and feeding
hydrolysates, respectively.