FIGURE 3.

Clavulanic acid (CA) liquid fermentation titers of F613-1, ΔcagRS and the complemented strain. Analysis of the change of CA concentration during fermentation (A,B). For detecting CA production during the liquid fermentation process, spores (10⁶/mL) were inoculated into 100 mL SCZ seed medium and cultured at 25°C, 200 rpm for 48 h to obtain seed cultures. Next, 5 mL seed cultures were transferred to 100 mL SCF fermentation medium supplemented with (A) or without (B) 1.6% (v/v) glycerol trioleate, and then grown at 25°C, 200 rpm for 216 h. To analyze CA concentration, 1 mL samples of fermentation liquid were collected at 24, 72, 120, 168, and 216 h, and centrifuged at 12000 rpm, 4°C, 5 min to collect the supernatant. Next, HPLC was performed immediately after the supernatant was filtered with a 0.22 mm filter membrane. (C,D) Growth curves of F613-1, ΔcagRS and ΔcagRScom with (C) or without (D) glycerol trioleate. Samples for growth curve analysis were harvested at seven time points (24, 48, 72, 96, 120, 168, and 216 h). Data are the mean ± SD of three separate experiments.