Process development of fuel ethanol production from lignocellulosic sugars using genetically engineered yeasts

Glucose and xylose are the major fermentable substrates present in lignocellulosic biomass, a potential feedstock for the commercial fuel ethanol production. Past research in this area has indicated that xylose fermentation and ethanol tolerance of the fermenting microorganism are major bottlenecks in the design of an economical fuel ethanol production process. The development of xylose fermenting yeasts by genetic engineering has potentially overcome the bottleneck of xylose conversion to ethanol. This dissertation deals with the fermentation process development of fuel ethanol production using a genetically engineered Saccharomyces yeast 1400 (pLNH33). Experiments indicate that along with a high ethanol tolerance of 13.6% (w/v), this yeast also ferments glucose and xylose simultaneously to ethanol. Kinetic studies indicate that ethanol is a major inhibitor in the process, and inhibits xylose fermentation more severely than glucose fermentation. In order to overcome the ethanol inhibition, simultaneous fermentation and ethanol recovery using $\rm CO\sb2$ stripping was performed in an energy efficient air-lift loop fermentor with side-arm (ALSA). This integrated process configuration significantly improves the ethanol productivity and yield from glucose and xylose. Acetic acid was also determined to be a fermentation inhibitor. An alkaline pretreatment method, that removes acetate and lignin from biomass, was applied for efficient ethanol production from corn cob (a model feedstock). Genetic stability of the recombinant yeast was investigated for process scale up. These studies indicate that xylose can be used as a selection pressure to maintain the cell in the recombinant state. This avoids the use of expensive antibiotics in the production process. On the other hand, fermentation media containing glucose was found to be non-selective. Results from kinetic studies were used for estimating parameters of an unstructured fermentation model that incorporates the effects of substrate inhibition, product inhibition, plasmid stability and inoculum size. Good agreements were obtained between the model predicted results and experimental data. Fermentation process monitoring was accomplished by using membrane introduction mass spectrometry (MIMS) coupled with flow injection analysis (FIA). An automated on-line monitoring and feedback control system was developed for rapid determination and control of the fermentation variables