Ethanol production depends on fermentation of simple sugars by microorganisms. The yield of potentially fermentable sugars from the conversion process is the critical response variable in assessing the value of alfalfa as an ethanol production feedstock. Potentially fermentable sugar yield is a function of both carbohydrate composition and concentration (discussed earlier), and the efficiency with which the cell wall polysaccharides are converted to simple sugars through processing. The results of two pretreatment methods have been reported previously. Ferrer et al. (2002) described parameters of ammonia processing of whole dried alfalfa hay that influenced the susceptibility of the fiber to subsequent enzymatic hydrol­ysis. The ammonia loading, moisture, time and temperature of treatment were varied and then the treated material digested with a mixture of cellulase, cello — biase, and xylanase. Conditions of 2 g ammonia g-1 DM, with 30% moisture and processing at 85°C for five minutes was shown to convert 76% of the theoretical yield of reducing sugars in the fiber. Approximately 200 mg sugars g-1 DM was obtained (Ferrer et al., 2002); however, the yield of ethanol produced from this material remains to be determined.

Liquid hot water (LHW) pretreatments of the fiber fraction obtained after wet fractionation of alfalfa have been optimized for maximum sugar conversion (Sreenath et al., 1999) and ethanol production (Sreenath et al., 2001). The LHW pretreatment was found to solubilize hemicellulose, and the resulting extract contained significant amounts of acetic acid and formic acid (Sreenath et al., 1999). The remaining fiber fraction (raffinate) when treated with cellulase released 59 g of reducing sugars from 100 g of substrate. Addition of dilute acid (0.07% sulfuric acid) to the LHW decreased the amount of reducing sugars released by cellulase treatment to 24 g 100 g-1 substrate (Sreenath et al., 1999). Fermentation of the raffinate fraction after LHW pretreatment was tested with two strains of Candida shehatae in a simultaneous saccharification and fermentation (SSF) process as well as a separate hydrolysis and fermentation (SHF) process (Sreenath et al., 2001). The yield of ethanol was 0.45 g ethanol g-1 sugar with SSF and 0.47g ethanol g-1 sugar with SHF. The extract from the LHW pretreatment was also used in fermentation experiments and was poorly fermented, most likely due to the presence of organic acids. Addition of dilute acid to the LHW treatment resulted in fractions that were poorly fermented. Although untreated fiber sub­strate was shown to yield 51 g reducing sugars from 100 g of substrate (Sreenath et al., 1999), the yield of ethanol by SHF and SSF was 0.25 and 0.16 g ethanol g-1 sugar, respectively (Sreenath et al., 2001). These experiments demonstrate the impact of pretreatment on saccharification and ethanol production as well as the requirement to optimize processes for each lignocellulosic feedstock.