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Consolidated bioprocessing (CBP) of lignocellulose to bioethanol refers to the combining of the four biological events required for this conversion process (production of saccharolytic enzymes, hydrolysis of the polysaccharides present in pretreated biomass, fermentation of hexose sugars, and fermentation of pentose sugars) in one reactor. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing. Although no natural microorganism exhibits all the features desired for CBP, a number of microorganisms, both bacteria and fungi, possess some of the desirable properties. This review focuses on progress made toward the development of baker's yeast (Saccharomyces cerevisiae) for CBP. The current status of saccharolytic enzyme (cellulases and hemicellulases) expression in S. cerevisiae to complement its natural fermentative ability is highlighted. Attention is also devoted to the challenges ahead to integrate all required enzymatic activities in an industrial S. cerevisiae strain(s) and the need for molecular and selection strategies pursuant to developing a yeast capable of CBP.

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Producing second-generation ethanol from the fi ve carbon (C5) sugars in bagasse and cane trash could increase ethanol yield at fi rst-generation sugarcane biorefi neries in Brazil. Co-fermenting C5 sugars with cane juice and molasses in the fi rst-generation fermenters is a potentially attractive process confi guration enabled by recent biotechnology advances. To assess the feasibility of this cofermentation, batch fermentations of molasses supplemented with xylose, the primary C5 sugar in hemicellulose, were conducted at lab scale using a yeast strain, M3799, engineered to ferment xylose. At cell loadings consistent with Brazilian fuel ethanol fermentation, M3799 was able to convert 17.8g/L of xylose sugar along with sugars typically found in molasses to produce 71.5g/L of ethanol in 8 h. Based on this capability, a process that produces a C5-enriched cane juice by integrating a steam pre-treatment reactor with the existing cane mill is investigated. Process modeling analysis of several integrated pre-treatment confi gurations predicts that sugar recovery on cane can be increased by 11% to 20% compared to traditional milling. This additional sugar can be fermented by C5 yeast in the fi rstgeneration fermentation to produce up to 37% more ethanol without effecting sugar coproduction. Due to close integration with the fi rst-generation host plant, these projects achieve a minimum ethanol selling price (MESP) of $0.17/L to $0.29/L at a 10% return, which is lower than the reported MESP for other second-generation projects and is cost competitive with fi rst-generation ethanol and gasoline. This translates to capital payback (EBITA/Capex) in two to four years.

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This study analyzes selection in continuous culture as a means to improve the growth of microorganisms dependent upon the expression of extracytoplasmic enzymes. A quantitative, theoretical model was developed that considers increases in enzyme activity and/or expression due to mutation in conjunction with reaction and diffusion at the cell surface and in the surrounding boundary layer. This model was applied to a system consisting of a recombinant yeast cell growing on either soluble or insoluble substrates by virtue of extracytoplasmic enzymes either with or without tethering to the cell surface. Our results indicate that selection of faster-growing cells can be effective, arbitrarily defined as a faster-growing mutant representing 1% of the population in < or =3 months, but only under some conditions. For both soluble and insoluble substrates, tethering of enzymes to the cell surface is required for selection to be effective under the conditions examined. Significant increases in heterologous enzyme expression (2.5-fold for mutants as compared to the parent strain) are also required. In the soluble substrate/enzyme tethered case, the value of k(S) must also be low in order for selection to be effective. Cells growing on non-native substrates by virtue of extracytoplasmic enzyme production are expected to experience selective pressure in response to several additional factors, including cell shape, distance of the cell-substrate gap, properties of the gap, and perhaps mutation frequency. However, these factors exert a smaller impact on selection time and it is not clear that favorable values for these factors are required in order for selection to be effective.