Production of ethanol from pulp mill hardwood and softwood spent sulfite liquors by genetically engineered E. coli

Abstract

Although lignocellulosic biomass and wastes are targeted as an attractive alternative fermentation feedstock for the production of fuel ethanol, cellulosic ethanol is not yet an industrial reality because of problems in bioconversion technologies relating both to depolymerization and fermentation. In the production of wood pulp by the sulfite process, about 50% of the wood (hemicellulose and lignin) is dissolved to produce cellulose pulp, and the pulp mill effluent ("spent sulfite liquor" SSL) represents the only lignocellulosic hydrolysate available today in large quantities (about 90 billion liters annually worldwide). Although softwoods have been the traditional feedstock for pulping operations, hardwood pulping is becoming more popular, and the pentose sugars in hardwood SSL (principally xylose) are not fermented by the yeasts currently being used in the production of ethanol from softwood SSL. This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinant Escherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30-35 g/L total reducing sugars). The pH was controlled at 7.0 to maximize tolerance to acetic acid. In contrast to the high-performance characteristics exhibited in synthetic media, formulated to mimic the composition of softwood and hardwood SSL (yield approaching theoretical maximum), performance in SSL media was variable with conversion efficiencies in the range of 67-84% for hardwood SSL and 53-76% for softwood SSL. Overlimiting treatment of HSSL, using Ca(OH)2, improved overall volumetric productivity two- to sevenfold to a max of 0.42 g/L/h at an initial cell loading of 0.5 g dry wt/L. A conversion efficiency of 92% (6.1 g/L ethanol) was achieved using diluted Ca(OH)2-treated hardwood SSL. The variable behavior of this particular genetic construct is viewed as a major detractant regarding its candidacy as a biocatalyst for SSL fermentations.