| Using existing infrastructures, the expanded use of ethanol could produce large amounts of liquid fuel for use in transportation. Carbohydrates found in lignocellulose (cellulose and hemicellulose) are an abundant and unused source of raw material (sugar) for producing commodity chemicals such as ethanol. Hemicellulosic sugars are easily hydrolyzed with dilute acids; however, the hydrolysis of cellulose requires more severe conditions, resulting in sugar degradation and the production of biologically inhibitory compounds. Hemicellulose sugars (particularly pentose sugars) are especially sensitive to acid degradation, and thus must be separated from cellulose before continued processing, adding to equipment costs. Alternatively, enzymes (cellulases) may be used to depolymerize cellulose without the problems associated with severe acid hydrolysis. The simultaneous saccharification and fermentation (SSF) of cellulose has previously been shown to greatly reduce the amount of cellulase needed for conversion to ethanol. Even so the costs of cellulases have been a major barrier to their large-scale use. The fermentation of hemicellulose combined with SSF (SSCF) provides further process simplification. Ethanologenic strains of Klebsiella oxytoca M5a1 have been shown to be exceptionally well suited for SSF. Previous studies have used expensive laboratory media, impractical for large scale use. Other work showed that in the presence of glucose, the use of the pentose sugars xylose and arabinose were repressed. This observation was attributed to the transcriptional regulation of the genes for xylose and/or arabinose metabolism by the cyclic-AMP receptor protein (CRP).; This study used K. oxytoca M5a1's natural ability to use urea as a nitrogen source to develop an inexpensive medium (containing corn steep liquor) to produce ethanol from lignocellulose. In glucose (9%) fermentation, using this new medium, elevated levels of products from the 2,3-butanediol pathway were produced. Deleting two genes exclusive to this pathway (budAB) greatly improved both ethanol productivity (30%) and yield (12%). In parallel work, it was found that over expression of a mutant CRP, CRP(in), insensitive to the normal intracellular signals, improved the metabolism of pentose sugars in the presence of glucose. Finally, genes encoding endoglucanase activities from Erwinia chrysanthemi (previously shown to be beneficial in SSF) were combined with other improvements to construct strains further improved for ethanol production from cellulose. The best strain (BW34) produced 38 g/L ethanol from 100 g/L cellulose (in SSF) compared to 28 g/L in strain P2, a 36% improvement. Ethanol yields were increased from 0.38 to 0.49 g ethanol per g cellulose in SSCF, which contained both xylose and cellulose (using 50% less fungal cellulose than in SSF). |
