Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth

Table 3 Lactate formation in flask fermentation

Strain	L- lactate (g/l)^a	D-lactate (g/l)^a	Yield of L-/D-lactate (%, w/w, based on total supplied glucose)
B0013-090B1	20.16 ± 0.90	0.02 ± 0.00	58
B0013-090B2	16.19 ± 0.92	0.03 ± 0.00	46
B0013-090B3	25.59 ± 1.08	0.03 ± 0.00	73
B0013-070	0.00 ± 0.00	27.27 ± 1.06	78
B0013-080C	0.00 ± 0.00	0.01 ± 0.00	/

^aThe flask fermentation experiments were carried out in 250 ml flasks with the working volume of 50 ml. Briefly, cells were grown in 50 ml of LB medium in a 250 ml flask at 37°C with shaking (200 rpm) for 8–10 h until cell density (OD₆₀₀) of 2.0-2.5 was reached. Cells were collected by centrifugation and resuspended in a modified M9 medium and then inoculated into 50 ml of the modified M9 medium complemented with 5 g/l glucose in a 250 ml flask with the initial cell density (OD₆₀₀) of 0.05 in triplicates. The cultivation was first carried out at 37°C and 200 rpm for 12 h, then 30 g/l glucose was added followed by stationary cultivation (anaerobic fermentation) for lactate formation at different temperatures. Calcium carbonate with a final concentration of 75 g/l was added for neutralization. Sampling was carried out during the cultivation. Lactic acid concentration was measured by HPLC equipped with UV (210 nm) and refractive index detectors, using a Shodex SH-1011 column (Shodex SH-1011 H610009; Showa Denko K.K., Kawasaki, Japan) with 0.01 M H₂SO₄ as eluent (0.6 ml/min; 50°C). Lactic acid isomeric purity was measured by HPLC using a chiral column (CLC-L; Advanced Separation Technologies Inc., Whippany, NJ, USA), at room temperature, equilibrated with 1 ml/min of 5 mM CuSO4 as the mobile phase and detected at 254 nm with a UV detector.

ISSN: 1475-2859