3.4 Cell Density, Viability, and Morphology in FBB
Cells in the fibrous matrices were collected from the FBB at the end of the continuous fermentation study and analyzed for their density, viability, and morphology. In general, more cells were observed at the lower parts closer to the entrance of the reactor with higher glucose and lower butanol concentrations since cell metabolism would be faster with higher concentrations of nutrients and lower concentrations of inhibiting end products. Biofilms with dense and thick layers of elongated rod-shaped cells covering the fibers in the fibrous matrices were observed throughout the fibrous bed, as can be seen in the scanning electron micrographs (Figure 7 ). The total cell density in the FBB was approx. 100 ± 15 g cell dry weight/L reactor with >70% cell viability. No obvious sporulation nor autolysis was observed even for cells from the top part of the bioreactor, suggesting that cells in the FBB remained healthy and were highly active throughout the entire process over a period of more than 400~600 h. It should be noted that there were continuous cell bleedings (~1.0 OD at 0.06 h-1and 0.12 h-1 and ~3.0 OD at 1.88 h-1) from the FBB throughout the continuous fermentation, which allowed for continuous removal of old or stressed cells to be replaced by new and more productive cells (Lewis and Yang, 1992). Furthermore, cells with the elongated morphology in the FBB were adapted to better tolerate toxic metabolites such as butyric acid and butanol. The elongated rod morphology with a higher specific surface area could increase mass transfer and efflux for exchanges of nutrients and metabolites between cells and the environment as previously reported (Suwannakham and Yang, 2005; Zhu and Yang, 2003). The beneficial effects of cell immobilization in the FBB including enhanced tolerance to toxic metabolic end products and increased product titer, productivity and yield have also been reported for other clostridia such as C. tyrobutyricum (Jiang et al., 2011) and C. formicoaceticum (Huang et al., 1998) and other bacteria such as propionibacteria (Zhang and Yang, 2009).