The work conducted in this project is an extension of the developments itemized in DE-FG-36-04GO14236. This program is designed to help the development of a biorefinery based around a raw sugar mill, which in Louisiana is an underutilized asset. Some technical questions were answered regarding the addition of a biomass to ethanol facility to existing sugar mills. The focus of this work is on developing technology to produce ethanol and valuable by-products from bagasse. Three major areas are addressed, feedstock storage, potential by-products and the technology for producing ethanol from dilute ammonia pre-treated bagasse. Sugar mills normally store bagasse in a simple pile. During the off season there is a natural degradation of the bagasse, due to the composting action of microorganisms in the pile. This has serious implications if bagasse must be stored to operate a bagasse/biorefinery for a 300+ day operating cycle. Deterioration of the fermentables in bagasse was found to be 6.5% per month, on pile storage. This indicates that long term storage of adequate amounts of bagasse for year-round operation is probably not feasible. Lignin from pretreatment seemed to offer a potential source of valuable by-products. Although a wide range of phenolic compounds were present inmore » the effluent from dilute ammonia pretreatment, the concentrations of each (except for benzoic acid) were too low to consider for extraction. The cellulosic hydrolysis system was modified to produce commercially recoverable quantities of cellobiose, which has a small but growing market in the food process industries. A spin-off of this led to the production of a specific oligosaccharide which appears to have both medical and commercial implications as a fungal growth inhibitor. An alternate use of sugars produced from biomass hydrolysis would be to produce succinic acid as a chemical feedstock for other conversions. An organism was developed which can do this bioconversion, but the economics of succinic acid production were such that it could not compete with current commercial practice. To allow recovery of commercial amounts of ethanol from bagasse fermentation, research was conducted on high solids loading fermentations (using S. cerevisiae) with commercial cellulase on pretreated material. A combination of SHF/SSF treatment with fed-batch operation allowed fermentation at 30% solids loading. Supplementation of the fermentation with a small amount of black-strap molasses had results beyond expectation. There was an enhancement of conversion as well as production of ethanol levels above 6.0% w/w, which is required both for efficient distillation as well as contaminant repression. The focus of fermentation development was only on converting the cellulose to ethanol, as this yeast is not capable of fermenting both glucose and xylose (from hemicellulose). In anticipation of the future development of such an organism, we screened the commercially available xylanases to find the optimum mix for conversion of both cellulose and hemicellulose. A different mixture than the spezyme/novozyme mix used in our fermentation research was found to be more efficient at converting both cellulose and hemicellulose. Efforts were made to select a mutant of Pichia stipitis for ability to co-ferment glucose and xylose to ethanol. New mutation technology was developed, but an appropriate mutant has not yet been isolated. The ability to convert to stillage from biomass fermentations were determined to be suitable for anaerobic degradation and methane production. An economic model of a current sugar factory was developed in order to provide a baseline for the cost/benefit analysis of adding cellulosic ethanol production.« less