FIG. 2.

Fed-batch cultivation of Z. denitrificans for PHB production from glycerol in the presence of NaCl (fed-batch 3). Z. denitrificans MW1 was cultivated in the presence of NaCl 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 fermenter was inoculated with 4% (vol/vol) 24-h preculture. The pH of the medium was controlled at 7.3 during the first 20 h of growth and then at 6.8 until the end of fermentation, using NaOH (4 N) or NH₄OH (25% [wt/vol]). The glycerol feeding solution (50% [vol/vol]) was supplemented with 1% MgSO₄ and two times the normal concentrations of trace elements, iron, and calcium in MSM. The pO₂ was controlled automatically at 20% saturation by adjusting aeration and agitation rates. Cells were grown for 63 h at 41°C. During the time course of cultivation, samples were withdrawn, and the concentrations of glycerol and ammonium, as well as the CDW and PHB content of the cells, were determined as described in Materials and Methods.