FIG. 3.

PHB production by Z. denitrificans MW1 during continuous feeding of glycerol (fed-batch 4). Cultivation was done in a Biostat UD-30 stirred-tank reactor containing 20 liters of MSM with glycerol as the sole carbon source. A total of 2% NaCl was added at the beginning of the fermentation. The bioreactor was inoculated with 4% (vol/vol) 24-h preculture. (A) Glycerol was fed continuously to keep its concentration higher than 10 g/liter. The glycerol feeding solution (80% [vol/vol]) was supplemented with MgSO₄ (1% [wt/vol]) and two times the normal concentrations of trace elements, iron, and calcium in MSM. During the time course of cultivation, samples were withdrawn, and the concentrations of glycerol and ammonium, as well as the CDW and PHB content of cells, were determined as described in Materials and Methods. (B) The following parameters were obtained by online monitoring: OD₈₅₀, pO₂ (controlled automatically at 20% saturation by adjusting aeration and agitation rates), and the pH of the medium (controlled at 7.3 during the first 20 h of growth and then at 6.8 to the end of fermentation, using NaOH [4 N] and NH₄OH [25% {wt/vol}] in parallel). Fermentation was operated for 50 h at 41°C. (C) Microscopic photographs showing the accumulation of PHB granules in cells during the entire time course of fermentation (9, 14, 24, 32, 45, and 50 h). The left panels (with size bars) show pictures obtained by phase-contrast light microscopy of cells possessing bright cytoplasmic inclusions. The right panels (without size bars) show fluorescence micrographs of PHB inclusions stained with Nile red.