| Several unique biocatalytic features of the thermophilic bacteria Clostridium thermocellum and Clostridium thermosaccharolyticum have encouraged their investigation for use in industrial fermentations. This study focuses on establishing the hydrolysis and fermentation characteristics of the cellulolytic Clostridium thermocellum in the context of ethanol production from cellulosic biomass. A newly developed defined growth medium is described and shown to support robust growth on fructose and cellobiose at concentrations up to 55 g/l. Batch cultivation of C. thermocellum on crystalline cellulose demonstrate that using the new medium, the organism hydrolyzes cellulose at essentially saturation rates for concentrations to 5%. Saturation rates are not obtained in well-mixed continuous cultures except at very low dilution rates, contrary to earlier studies. Characterization of C. thermocellum fermentation in batch and continuous culture, on simple as well as complex sugars, indicate the diversion of significant amounts of substrate carbon to a compound, identified in this study, as a small, secreted polysaccharide. The substrate diversion results in low ethanol yields, emphasizing the need to understand the basis of polysaccharide production and develop cultivation techniques which eliminate its accumulation. One such technique shown to be effective is co-cultivation of C. thermocellum with the pentose fermenter, Clostridium thermosaccharolyticum. In co-cultivation, polysaccharide production is not detected and a concomitant increase in yield of fermentation products is measured. Cultivated on the new medium, a batch co-culture of C. thermocellum and C. thermosaccharolyticum was able to ferment 100 g/l cellulose in 70 hrs. to produce 25 g/l ethanol. |
