2.3 Continuous Fermentation
Continuous fermentation was conducted with cells immobilized in a fibrous bed bioreactor, which was made of a water-jacketed glass column. The glass column with a working volume of 500 mL was packed with a spirally wound cotton towel laminated with a stainless-steel wire mesh as spacers between adjacent fibrous matrices (Figure 1 ). The FBB was autoclaved at 121 oC for 30 min, left overnight at room temperature to allow spore germination, and autoclaved again for one hour. The FBB was purged with N2 from the bottom of the bioreactor for ~1 h to remove air and then aseptically connected to a 10-L feed medium bottle containing sterile P2 medium. The bioreactor was then partially filled with the medium while continued being sparged with N2 for 10 min and inoculated with 60~100 mL of an overnight culture grown in a serum bottle. The FBB was controlled at 35 oC and held static for 12~24 h to allow cells to grow and attach/adsorb onto the fibrous bed. When significant gas production (bubbles) was observed and the fermentation broth had reached ~1.0 OD600, continuous fermentation was started by pumping the feed medium containing 50 g/L glucose and 4 g/L butyric acid into the bottom of the FBB at a constant dilution rate of 0.06 h-1. The continuous fermentation study was operated for over 28 days or until reaching a pseudo-steady state. The continuous fermentation was then studied in the same FBB fed with P2 medium containing 60 g/L glucose and 6.8 g/L butyric acid at 0.12 h-1 dilution rate. A smaller FBB with a 125-mL working volume was also set up for studying the continuous fermentation at a higher dilution rate of 1.88 h-1. Fermentation broth samples were taken periodically from the effluent stream throughout the continuous fermentation studies and analyzed for OD600and concentrations of glucose, butyric and acetic acids, and solvents (acetone, butanol, and ethanol). At the end of the continuous fermentation studies, the fibrous matrix with immobilized cells was removed from the FBB and examined for cell density, viability, and morphology.