Search

..................................

Select Level of Detail

At a Glance

General

Technical

............................

+ Ethanol Production - Ethanol from Cellulose Resources

............................

Access BioWeb Content

Search

Explore By Topic

Browse Index

............................

BioWeb Glossary

Search

Alphabetical Listing

............................

Contributors Log in

bioweb.sungrant.org » Technical » Biofuels » Technologies » Ethanol Production » Ethanol from Cellulose Resources

Ethanol from Cellulose Resources	Printer Friendly

Most of the fuel ethanol produced in the United States is made from corn (> 90%) (RFA, 2006). Most analysts agree that the ability to significantly increase ethanol production using corn grain is limited, and that large increases will require the use lignocellulose resources such as agricultural residues, grasses, and wood.   Ethanol is produced by fermenting plant carbohydrates with yeast. Plant carbohydrates are grouped as soluble sugars (such as sucrose from sugarcane), storage carbohydrates (such as starch from grains and tubers), and structural carbohydrates which make up the plant cell wall (such as cellulose, hemicellulose, and pectin). The principal carbohydrates contained in lignocellulose resources are the structural carbohydrates. These carbohydrates, along with proteins and lignin, form the complex matrix of plant cell walls that give plants structural stability and protection from the environment (Somerville, 2004).   Cellulose is a straight chained (linear) polymer of glucose molecules joined by ß (1-4) glycosidic bonds (figure 1) (Gardner, 1974). The multiple hydroxyl groups (OH) situated along a cellulose chain bond with the hydroxyl groups of other cellulose chains to form tight crystalline structures (microfibrils). Microfibrils have high tensile strength (resistance to being pulled apart, e.g. a rope) and are the major structural components of all plant cell walls (McCann, 2001). Cellulose is far more abundant in nature than is starch, but the high tensile strength and chemical stability of cellulose make it much more difficult to break down into glucose molecules, making the process of converting cellulose to ethanol more complex than for starch.          In addition to cellulose, plant cell walls contain significant quantities of hemicellulose. Hemicellulose is a heterogeneous polymer composed of a backbone comprised principally of pentose (5 carbon) sugars (figure 2). Xylose is the principal pentose found in grasses and hardwood tree species (up to 40% of the sugars), while mannose is the major pentose in softwood tree species (Somerville, 2004). Other sugars (glucose, galactose, arabinose, or rhamnose) and organic acids (acetic, glucuronic, ferulic and p-coumaric acids) can substitute for some of the xylose and mannose sugars in the hemicellulose backbone.                                 Producing fuel ethanol from lignocellulose resources involves four major steps - preparation of the feedstock (pretreatment and hydrolysis), fermentation of the sugars, recovery of the ethanol, and handling of the coproducts.

Feedstock preparation
The stability and chemical complexity of cellulose increase the difficulty of breaking it down into glucose, a situation frequently referred to as the recalcintrance of cellulose. A number of pretreatment approaches are being explored to overcome this problem. Pretreatment is the general term used to describe the processing steps preceding hydrolysis of cellulose and hemicellulose into fermentable sugars. The goal of any pretreatment technology is to alter or remove structural and compositional factors present in plant biomass that hinder the breakdown (hydrolysis) of cell wall polysaccharides (polymers of simple sugars) into the fermentable simple sugars (Mosier, 2005a, 2005b; Grohmann, 1984; Lynd, 1999; McMillan, 1994). Pretreatment methods often involve harsh conditions and non-selectively hydrolyze the polysaccharides in the plant material. Sugars that are produced during pretreatment are also subject to further degradation to form compounds that act as inhibitors to the subsequent fermentation process (Klinke, 2004; Olsson, 1996; Palmqvist, 2000; Taherzadeh, 1997).    Pretreatment methods are usually either physical or chemical, although some approaches incorporate both (Hsu, 1996; McMillan, 1994). Chemical approaches use acids or bases to promote the hydrolysis of cellulose by removing the hemicellulose and/or lignin during pretreatment. The most commonly used acids and bases are sulfuric acid (H2SO4), sodium hydroxide (NaOH) or ammonia. Other chemicals used include cellulose solvents such as alkaline H2O2, ozone, organosolv (Lewis acids, FeCl3, and Al2SO4 in aqueous alcohol), glycerol, dioxane, phenol, or ethylene glycol, which disrupt the cellulose structure and promote hydrolysis (Wood, 1988).   Dilute sulfuric acid is commonly used as a pretreatment method, but increases cost, due to the need for reactors constructed of expensive steel materials, and results in the formation of unwanted salts that require neutralizing agents (Hinman, 1992; Lynd, 1996; Lynd, 1999;  Thompson, 1979). Liquid hot water pretreatment with pH control effectively dissolves hemicellulose and lignin, while minimizing degradation of monosaccharides without the need for costly and potentially dangerous pretreatments and neutralizing agents (Kim, 2005; Weil, 1998). Other pretreatment methods include steam explosion (Abatzoglou, 1992; Heitz, 1991; Ramos, 1992), ammonia fiber expansion (AFEX) (Gollapalli, 2002; Teymouri, 2005; Wang, 1998), and other chemical solvents (Hsu, 1996; McMillan, 1994). Few of these pretreatment processes have been fully commercialized or tested at industrial scales (Mosier, 2005a, 2005b). Scaling pretreatment processes to commercial sizes and the associated reactor design issues remains a major barrier to the commercial production of fuel ethanol from lignocellulosic biomass (Lynd, 1999; Mosier, 2005a, 2005b). A summary and evaluation of a number of pretreatment processes can be found in Lynd (1999), Mosier (2005a, 2005b), and Kim (2005).   Following pretreatment, plant cell wall polysaccharides are more susceptible to chemical or enzymatic hydrolysis that breaks them into monomeric (single) sugars (saccharification) that can be fermented into ethanol (Lynd, 1999). Depending on the type and effectiveness of the pretreatment method, hydrolysis takes 24-48 hours to complete (Lin, 2006; Olsson, 1996). In an effort to reduce the overall time needed to produce ethanol, the hydrolysis and fermentation processes are being combined, rather than conducted sequentially, in a process called simultaneous saccharification and fermentation (SSF) (Lynd, 1999). In SSF, the fermenting microorganism (e.g., yeast) and enzymes that hydrolyze the polysaccharides are added at the same time so that the sugars are fermented as soon as they are available in a just-in-time process. SSF reduces ethanol production time and reduces the amount of enzyme used when a separate hydrolysis step is conducted, due to the formation of fewer compounds that inhibit ethanol production (Lin, 2006).

Fermentation of sugars
Fermentation involves microorganisms which consume sugars as a food source. Ethanol fermentation results in four major products: additional yeast cells (cell division), ethanol, carbon dioxide, and heat. One molecule of glucose will yield, stoichiometrically, 2 molecules of ethanol plus 2 molecules of carbon dioxide (Figure 3). On a mass basis, one kilogram of glucose will theoretically produce 0.51 kilogram of ethanol and 0.49 kilogram of carbon dioxide. However, glucose consumed to generate additional yeast cells (cell mass) does not result in the production of ethanol. Most industrial fermentation processes operate at 90–95% of the theoretical yield of ethanol from glucose fed to the yeast.                      The fermentation process for producing fuel ethanol from cellulose is similar to that from corn grain. The fermentation media contains insoluble solids from the plant biomass, yeast, and soluble products from the pretreatment and saccharification steps. Hexoses (the six carbon sugars glucose and galactose) produced by hydrolysis of cellulose and hemicellulose can be fermented to ethanol using existing industrial strains of Saccharomyces cerevisiae.   The major difference between using cellulosic feedstocks and starch feedstocks lies in the existence of relatively large amounts of pentose sugars (five carbon sugars such as xylose and arabinose) contained in the hemicellulose. These sugars also need to be fermented to make the overall process economically feasible (Eggeman, 2005; Nagle, 1999). Existing strains of S. cerevisiae cannot directly ferment xylose, but can ferment xylulose through the pentose phosphate pathway (figure 4). Other yeasts and bacteria can ferment xylose or xylitol; efforts utilizing the tools of biotechnology are underway to develop industrial microorganisms capable of efficiently converting xylose to ethanol. A number of approaches are being explored.              S. cerevisiae can ferment xylulose, but lacks the necessary enzymes to convert xylose to xylulose. Early efforts to metabolically engineer S. cerevisiae to be able to ferment xylose focused on inserting a bacterial gene (xylose isomerase XI) capable of converting xylose to xylulose (Ho, 1983a, 1983b; Morgan, 1983). However, the resulting genetically modified yeast strain was not very efficient in fermenting xylose to ethanol. Later attempts focused on inserting genes obtained from yeast species that were more closely related to S. cerevisiae. Successful cloning and over-expression of the yeast xylulokinase (XK) gene in S. cerevisiae was completed in 1987 (Ho, 1989; Xue, 1990) and resulted in a strain that could effectively co-ferment both glucose and xylose. This was accomplished by inserting copies of xylose reductase (XR) and xylitol dehydrogenase (XD) genes from Pichia stipitis, and the xylulokinase (XK) gene, along with the proper promoter sequences into a plasmid which was then used to genetically transform S. cerevisiae. Improvements in this approach have resulted in the development of more than 10 different yeast strains (e.g., 1400(LNH-ST); 1400(pLNH32); 424A (LNH-ST)) that are stable and able to convert glucose and xylose to ethanol (Ho, 1999; Ho, 1998; Ho, 2000; Toon, 1997). The 424A (LNH-ST) strain is being evaluated by companies to ferment cellulosic biomass resources (Corrington, 2003; Dennison, 2003; Helle, 2004). Four other groups (one each in Sweden, Finland, Germany, and Japan) have also genetically engineered S. cerevisiae to ferment xylose using the XR and XD genes from P. stipitis (Kotter, 1990; Tantirungkij, 1993; Walfridsson, 1997). Additional efforts are underway to improve the tolerance of S. cerevisiae to inhibitory compounds found in lignocellulose or generated during processing (Liu, 2004; Olsson, 1996; Palmqvist, 2000; Zaldivar, 2001).   The use of microorganisms other than S. cerevisiae to ferment xylose, such as the bacteria Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis is also being explored. E. coli and K. oxytoca are able to metabolize several sugars, but are not efficient producers of ethanol. Z. mobilis is an efficient producer of ethanol, but can only utilize glucose and fructose. All three bacteria have been successfully genetically modified to produce ethanol from xylose (Dien, 2003; Zaldivar, 2001).   E. coli has been extensively studied, is easily cultured, and is used commercially to produce recombinant proteins. It can ferment a wide array of sugars without the need for complex growth factors or nutrients. However, it is less tolerant of harsh conditions (heat, salt, shear, acid, etc.) than yeast (Dien, 2003). Genetic modification of E. coli has resulted in the production of the strains K011 and FBR5 which have been shown to ferment sugars derived from a number of biomass resources (pine, sugarcane bagasse, corn stover, and corn hulls and germ meal) (Ingram, 1987; Asghari, 1996; Barbosa, 1992; Dien, 1997; Dien, 2000). Further effort is needed to improve the tolerance of E. coli to high levels of ethanol in order to increase ethanol yields (Gonzalez, 2003).   Z. mobilis has several properties that make it appealing, including a native homo-ethanol fermentation metabolism, tolerance to high ethanol concentrations (up to 12% by weight), high yield and productivity of ethanol, and is generally regarded as safe (GRAS) (Dien, 2003; Zaldivar, 2001). However, Z. mobilis can only ferment glucose and fructose to ethanol. It has been successfully modified to ferment xylose to ethanol through the incorporation of genes from E. coli (xylose isomerase, xylulose kinase, transketolase, and transaldolase), which produced a strain capable of co-fermenting glucose and xylose (Zhang, 1995). A similar strategy was employed to engineer a strain capable of fermenting arabinose to ethanol (Deanda, 1996). These initial efforts have led to the development of Z. mobilis AX101 which carries within its chromosomal DNA, all of the genes needed to ferment xylose, arabinose, and glucose (Mohagheghi, 2002; Mohagheghi, 1998). Integration of the genes into the chromosome eliminates the need to use antibiotics to maintain exogenous DNA carried on plasmids (Dien, 2003). Additional efforts are underway to improve the tolerance of Z. mobilis to acetic acid, an organic acid naturally found in lignocellulose (Mohagheghi, 2002; Tao, 2005).   Efforts are underway to improve the industrial performance of microorganisms to ferment sugars to ethanol. Efforts include improving the efficiency by which sugars are converted to ethanol (i.e., yields of 90% of theoretical yields are needed) and increasing the rate of productivity (i.e., the rate at which ethanol is produced to be > 1 g L-1 hr-1) (Zaldivar, 2001; Dien, 2003). Additionally, the microorganisms must tolerate high ethanol levels (> 4 wt%) to make distillation and ethanol recovery practical (Ladisch, 1979). The microorganism must also be resistant to fermentation inhibiting compounds (e.g., acetic acid, furfural, and lignin degradation compounds), and be tolerant of acid conditions (pH 4 – 5) (Palmqvist, 1999; Palmqvist, 2000).

Ethanol recovery
The recovery of ethanol produced in cellulosic processes is expected to use technology similar to that used in corn ethanol or sugar cane ethanol facilities. These technologies include repeated distillation (vaporization) and condensation of the ethanol-water mixtures produced during fermentation until the azeotrope point is reached (the point at which the difference between the boiling points of water and ethanol ceases to exist, which occurs when the mixture is 95.6% ethanol and 4.4% water by weight). At this point, the ethanol and water both vaporize to the same extent and cannot be further fractionated by distillation and final purification will by performed by the use of molecular sieves (zeolites which adsorb water from a vapor/gas mixture) (Al-Asheh, 2004; Kwiatkowski, 2006; Ladisch, 1979; Wankat, 1988).   Co-product production and handling   Unlike processes that produce ethanol from corn, the residual solid material produced in lignocellulose processes has little value as an animal feed as it is high in lignin while low in fiber and protein (McAloon, 2000). The material can be used to produce heat, steam, and electricity needed to run the ethanol facility, with the excess electricity sold to the electrical grid (Nagle, 1999).

References
Abatzoglou, N.; Chornet, E.; Belkacemi, K.; and Overend, R. P. (1992). Phenomenological Kinetics of Complex-Systems - the Development of a Generalized Severity Parameter and Its Application to Lignocellulosics Fractionation. Chemical Engineering Science, 47(5), 1109-1122. Al-Asheh, S.; Banat, F.; and Al-Lagtah, N. (2004). Separation of ethanol-water mixtures using molecular sieves and biobased adsorbents. Chemical Engineering Research and Design, 82(A7), 855-864. Asghari, A.; Bothast, R. J.; Doran, J. B.; and Ingram, L. O. (1996). Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11. Journal of Industrial Microbiology, 16(1), 42-47. Barbosa, M. D. S.; Beck, M. J.; Fein, J. E.; Potts, D.; and Ingram, L. O. (1992). Efficient Fermentation of Pinus Sp Acid Hydrolysates by an Ethanologenic Strain of Escherichia-Coli. Applied and Environmental Microbiology, 58(4), 1382-1384. Corrington, P. and Abbas, C. (2003). Screening ethanoglogens on corn fiber hydrolysate. Paper presented at the 25th  Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colorado, USA. Deanda, K.; Zhang, M.; Eddy, C.; and Picataggio, S. (1996). Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Applied and Environmental Microbiology, 62(12), 4465-4470. Dennison, E. and Abbas, C. (2003). Evaluation of recombinant microorganism ethanol fermentation of corn fiber hydrolysate. Paper presented at the 25th  Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colorado, USA. Dien, B. S.; Cotta, M. A. and Jeffries, T. W. (2003). Bacteria engineered for fuel ethanol production: current status. Applied Microbiology and Biotechnology, 63(3), 258-266. Dien, B. S.; Hespell, R. B.; Ingram, L. O. and Bothast, R. J. (1997). Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40. World Journal of Microbiology and Biotechnology, 13(6), 619-625. Dien, B. S.; Nichols, N. N.; O'Bryan, P. J. and Bothast, R. J. (2000). Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Applied Biochemistry and Biotechnology, 84-6, 181-196. Eggeman, T. and Elander, R. T. (2005). Process and economic analysis of pretreatment technologies. Bioresource Technology, 96(18), 2019-2025. Gardner, K. H. and Blackwel, J. (1974). Structure of Native Cellulose. Biopolymers, 13(10), 1975-2001. Gollapalli, L. E.; Dale, B. E.; and Rivers, D. M. (2002). Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw. Applied Biochemistry and Biotechnology, 98, 23-35. Gonzalez, R.; Tao, H.; Purvis, J. E.; York, S. W.; Shanmugam, K. T.; and Ingram, L. O. (2003). Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: Comparison of KO11 (Parent) to LY01 (resistant mutant). Biotechnology Progress, 19(2), 612-623. Grohmann, K.; Himmel, M.; Rivard, C.; Tucker, M.; Baker, J.; Torget, R.; et al. (1984). Chemical-Mechanical Methods for the Enhanced Utilization of Straw. Biotechnology and Bioengineering, 137-157. Heitz, M.; Capekmenard, E.; Koeberle, P. G.; Gagne, J.; Chornet, E.; Overend, R. P.; et al. (1991). Fractionation of Populus-Tremuloides at the Pilot-Plant Scale - Optimization of Steam Pretreatment Conditions Using the Stake-Ii Technology. Bioresource Technology, 35(1), 23-32. Helle, S. S.; Murray, A.; Lam, J.; Cameron, D. R.; and Duff, S. J. B. (2004). Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor. Bioresource Technology, 92(2), 163-171. Hinman, N. D.; Schell, D. J.; Riley, C. J.; Bergeron, P. W.; and Walter, P. J. (1992). Preliminary Estimate of the Cost of Ethanol-Production for Ssf Technology. Applied Biochemistry and Biotechnology, 34-5, 639-649. Ho, N.; Chen, Z. D.; Brainard, A. P.; and Sedlak, M. (1999). Successful Design and Development of Genetically Engineered Saccharomyces Yeasts for Effective cofermentation of Glucose and Xylose from cellulosic Biomass to Fuel ethanol. In G. T. Tsao (Ed.), Advances in Biochemical Engineering/Biotechnology. Berlin, Heidelberg: Springer-Verlag. Ho, N. and Tsao, G. T. (1998). Recombinant Yeasts for Effective Fermentation of Glucose and Xylose. United States. Ho, N. W. Y. and Chang, S. F. (1989). Cloning of Yeast Xylulokinase Gene by Complementation of Escherichia-Coli and Yeast Mutations. Enzyme and Microbial Technology, 11(7), 417-421. Ho, N. W. Y.; Chen, Z. D. and Sedlak, M. (2000). Strategies for successful metabolic engineering of Saccharomyces yeasts to effectively co-utilize glucose and xylose from renewable cellulosic biomass for the production of ethanol and other industrial products. Abstracts of Papers of the American Chemical Society, 219, U178-U178. Ho, N. W. Y.; Rosenfeld, S.; and Stevis, P. (1983). Cloning of the Escherichia-Coli Xylose Isomerase Gene in Yeast. Federation Proceedings, 42(7), 2167-2167. Ho, N. W. Y.; Stevis, P.; Rosenfeld, S.; Huang, J. J. and Tsao, G. T. (1983). Expression of the Escherichia-Coli Xylose Isomerase Gene by a Yeast Promoter. Biotechnology and Bioengineering, 245-250. Hsu, T.-A. (1996). Pretreatment of biomass. In C. Wyman (Ed.), Handbook on Bioethanol: Production and Utilization (pp. 179-195). Washington: Taylor and Francis. Ingram, L. O.; Conway, T.; Clark, D. P.; Sewell, G. W. and Preston, J. F. (1987). Genetic-Engineering of Ethanol-Production in Escherichia-Coli. Applied and Environmental Microbiology, 53(10), 2420-2425. Kim, Y.; Hendrickson, R.; Mosier, N.; and Ladisch, M. R. (2005). Plug-flow reactor for continuous hydrolysis of glucans and xylans from pretreated corn fiber. Energy and Fuels, 19(5), 2189-2200. Klinke, H. B.; Thomsen, A. B.; and Ahring, B. K. (2004). Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Applied Microbiology and Biotechnology, 66(1), 10-26. Kwiatkowski, J. R.; Mcaloon, A. J.; Taylor, F.; and Johnston, D. B. (2006). Modeling the Process and Costs of Fuel Ethanol Production by the Corn Dry-Grind Process. Industrial Crops and Products, 23, 288-296. Ladisch, M. R. and Dyck, K. (1979). Dehydration of Ethanol: New Approach Gives Positive Energy Balance. Science, 205, 898-900. Lin, Y. and Tanaka, S. (2006). Ethanol fermentation from biomass resources: current state and prospects. Applied Microbiology and Biotechnology, 69(6), 627-642. Liu, Z. L; Dien, B. S.; Berhow, M. A.; Kurtzman, C. P. and Gorsich, S. W. (2004). Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethlfuran. Journal of Industrial Microbiology and Biotechnology, 31(8), 345-352. Lynd, L. R. (1996). Overview and evaluation of fuel ethanol from cellulosic biomass: Technology, economics, the environment, and policy. Annual Review of Energy and the Environment, 21, 403-465. Lynd, L. R.; Wyman, C. E., and Gerngross, T. U. (1999). Biocommodity engineering. Biotechnology Progress, 15(5), 777-793. McAloon, A.; Tayor, F.; Yee, W.; Ibsen, K., and Wooley, R. (2000). Determining the Cost of Producing Ethanol from Corn Starch and Lignocellulosic Feedstocks. NREL/TP-580-28893. McCann, M. C.; Bush, M.; Milioni, D.; Sado, P.; Stacey, N. J.; Catchpole, G. et al. (2001). Approaches to understanding the functional architecture of the plant cell wall. Phytochemistry, 57(6), 811-821. McMillan, J. D. (1994). Pretreatment of Lignocellulosic Biomass. In Enzymatic Conversion of Biomass for Fuels Production (Vol. 566, pp. 292-324). Mohagheghi, A.; Evans, K.; Chou, Y. C. and Zhang, M. (2002). Cofermentation of glucose, xylose, and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101. Applied Biochemistry and Biotechnology, 98, 885-898. Mohagheghi, A.; Evans, K.; Finkelstein, M. and Zhang, M. (1998). Cofermentation of glucose, xylose, and arabinose by mixed cultures of two genetically engineered Zymomonas mobilis strains. Applied Biochemistry and Biotechnology, 70-2, 285-299. Morgan, A. J. and Nicolaidis, A. (1983). Cloning of Bacterial D-Xylose Isomerase in Yeast. Heredity, 51(OCT), 524-524. Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Lee, Y. Y.; Holtzapple, M., et al. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96(6), 673-686. Mosier, N. S.; Hendrickson, R.; Brewer, M; Ho, N.; Sedlak, M.; Dreshel, R., et al. (2005). Industrial scale-up of pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production. Applied Biochemistry and Biotechnology, 125(2), 77-97. Nagle, N.; Ibsen, K.; and Jennings, E. (1999). A process economic approach to develop a dilute-acid cellulose hydrolysis process to produce ethanol from biomass. Applied Biochemistry and Biotechnology, 77-9, 595-607. Olsson, L. and HahnHagerdal, B. (1996). Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme and Microbial Technology, 18(5), 312-331. Palmqvist, E.; Grage, H.; Meinander, N. Q.; and Hahn-Hagerdal, B. (1999). Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnology and Bioengineering, 63(1), 46-55. Palmqvist, E. and Hahn-Hagerdal, B. (2000). Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource Technology, 74(1), 25-33. Ramos, L. P.; Breuil, C.; and Saddler, J. N. (1992). Comparison of Steam Pretreatment of Eucalyptus, Aspen, and Spruce Wood Chips and Their Enzymatic-Hydrolysis. Applied Biochemistry and Biotechnology, 34-5, 37-48. Somerville, C.; Bauer, S.; Brininstool, G.; Facette, M.; Hamann, T.; Milne, J., et al. (2004). Toward a systems approach to understanding plant-cell walls. Science, 306(5705), 2206-2211. Taherzadeh, M. J.; Eklund, R.; Gustafsson, L.; Niklasson, C.;and Liden, G. (1997). Characterization and fermentation of dilute-acid hydrolyzates from wood. Industrial and Engineering Chemistry Research, 36(11), 4659-4665. Tao, F.; Miao, J. Y.; Shi, G. Y. and Zhang, K. C. (2005). Ethanol fermentation by an acid-tolerant Zymomonas mobilis under non-sterilized condition. Process Biochemistry, 40(1), 183-187. Teymouri, F.; Laureano-Perez, L.; Alizadeh, H., and Dale, B. E. (2005). Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Bioresource Technology, 96(18), 2014-2018. Thompson, D. R. and Grethlein, H. E. (1979). Design and Evaluation of a Plug Flow Reactor for Acid-Hydrolysis of Cellulose. Industrial and Engineering Chemistry Product Research and Development, 18(3), 166-169. Toon, S. T.; Philippidis, G. P.; Ho, N. W. Y.; Chen, Z. D.; Brainard, A.; Lumpkin, R. E.; et al. (1997). Enhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modes. Applied Biochemistry and Biotechnology, 63-5, 243-255. Wang, L.; Dale, B. E.; Yurttas, L.; and Goldwasser, I. (1998). Cost estimates and sensitivity analyses for the ammonia fiber explosion process. Applied Biochemistry and Biotechnology, 70-2, 51-66. Wankat, P. C. (1988). Introduction to Column Distillation. In Equilibrium-Staged Separations. New York, NY: Prentice Hall PTR. Weil, J.; Brewer, M.; Hendrickson, R.; Sarikaya, A.v and Ladisch, M. R. (1998). Continuous pH monitoring during pretreatment of yellow poplar wood sawdust by pressure cooking in water. Applied Biochemistry and Biotechnology, 70-2, 99-111. Wood, T. M. and Saddler, J. N. (1988). Increasing the Availability of Cellulose in Biomass Materials. Methods in Enzymology, 160, 3-11. Xue, X. D., and Ho, N. W. Y. (1990). Xylulokinase Activity in Various Yeasts Including Saccharomyces-Cerevisiae Containing the Cloned Xylulokinase Gene. Applied Biochemistry and Biotechnology, 24-5, 193-199. Zaldivar, J.; Nielsen, J.; and Olsson, L. (2001). Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Applied Microbiology and Biotechnology, 56(1-2), 17-34. Zhang, M.; Franden, M. A.; Newman, M.; McMillan, J.; Finkelstein, M.; and Picataggio, S. (1995). Promising Ethanologens for Xylose Fermentation - Scientific Note. Applied Biochemistry and Biotechnology, 51-2, 527-536.