2.3. Culture conditions for mixed acids production
Microbial strains precultured in Reinforced Clostridial Medium
(10 g/L peptone, 10 g/L beef extract, 3 g/L yeast extract, 5 g/L
dextrose, 5 g/L sodium chloride, 1 g/L soluble starch, 3 g/L sodium
acetate, 0.5 g/L cysteine$HCl) were transferred to base medium
containing 9.2 g/L G. amansii and 0.2 g CaCO3 for mixed acid
fermentation. For fermentation experiments, 2-L reactor volumes
in a 5-L stirred-vessel system (Kobiotech. Co. Ltd) supplied with
nitrogen gas (0.15 vvm) were fermented at 37 C with shaking
(120 rpm). The pH was maintained at 6.5e7.0 using 2N HCl or 2 M
NaOH. Iodoform, added individually to each reactor periodically
throughout the fermentation process from stock solutions (20 g/L)
in ethanol, was used as a methanogen inhibitor in all experiments.