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Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion to ethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not fermentable to ethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galacturonic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper units of cellulase, 120.4 polygalacturonase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g(-1) acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L(-1)), a combination of 20 FPU cellulase g(-1) bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L(-1). Alternatively, almost 40 g ethanol L(-1) was produced with 10 FPU cellulase g(-1) bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU(-1) of commercial cellulase. (c) 1994 John Wiley & Sons, Inc.

Complete enzymatic hydrolysis of cellulose to glucose is generally required for efficient fermentation to ethanol. This hydrolysis requires endoglucanase, exoglucanase, and cellobiase. The Gram‐negative bacterium, Klebsiella oxytoca, contains the native ability to transport and metabolize cellobiose, minimizing the need for extracellular cellobiase. Strain P2 is a recombinant derivative in which the Zymomonas mobilis pdc and adhB genes have been integrated into the chromosome and expressed, directing the metabolism of pyruvate to ethanol. This organism has been evaluated in simultaneous sacchari‐fication and fermentation (SSF) experiments to determine optimal conditions and limits of performance. The temperature was varied between 32 and 40 °C over a pH range of 5.0–5.8 with 100 g/L crystalline cellulose (Sigmacell 50, Sigma Chemical Company, St. Louis, MO) as the substrate and commercial cellulase (Spezyme CE, South San Francisco, CA). A broad optimum for SSF was observed, with a pH of 5.2–5.5 and temperatures of 32–35 °C, which allowed the production of over 44 g of ethanol/L (81–86% of the maximum theoretical yield). Although the rate of ethanol production increased with cellulase, diminishing improvements were observed at enzyme loadings above 10 filter paper units/g of cellulose. Over 40 g of ethanol/L was produced with relatively low enzyme loadings: 7–10 filter paper units/g of cellulose. Two optimal SSF conditions were identified for fermentation yield with strain P2: pH 5.2 at 35 °C and pH 5.5 at 32 °C. Under these conditions, 47 g of ethanol/L was produced in 144 h (0.48 g of ethanol/g of cellulose). Maximal rates of ethanol production were observed at 37 °C and pH 5.0 and produced over 40 g of ethanol/L in 72 h (final yield of 0.432 g of ethanol/g of cellulose after 96 h). All fermentations except those conducted at 40 °C and low pH (pH 5.0–5.5) exceeded 70% of theoretical ethanol yields. Tight process controls may not be required using this organism since temperatures between 32 and 37 °C at pH ranges between 5.0 and 5.8 still produced good yields.

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