Enhancement of Cellulose Saccharification Kinetics Using an Ionic Liquid Pretreatment Step

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1 Enhancement of Cellulose Saccharification Kinetics Using an Ionic Liquid Pretreatment Step Anantharam P. Dadi, Sasidhar Varanasi, Constance A. Schall Department of Chemical and Environmental Engineering, The University of Toledo, Toledo, Ohio 43606; telephone: ; fax: ; Received 28 October 2005; accepted 23 May 2006 Published online 17 August 2006 in Wiley InterScience ( DOI: /bit Abstract: Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay. ß 2006 Wiley Periodicals, Inc. Keywords: cellulose; hydrolysis; saccharification; ionic liquids; pretreatment INTRODUCTION Cellulose is an abundant renewable polymer which constitutes a large fraction of the lignocellulosic material found in plant cell walls (30 50%). It is a highly crystalline polymer of D-anhydroglucopyranose units joined together in long chains by b-1,4 glycosidic bonds (Lynd et al., 1999, 2002; Mansfield et al., 1999). The individual cellulose chains are joined by a network of inter- and intra-molecular hydrogen bonding and van der Waals forces (Pizzi and Eaton, 1985). The high crystallinity of cellulose makes it recalcitrant to hydrolysis into its individual glucose subunits. This highly ordered structure of cellulose has to be overcome in order to make the hydrolysis process viable for producing simple sugars for fermentation to produce ethanol fuel and other biobased products. Correspondence to: C.A. Schall Contract grant sponsor: National Science Foundation Contract grant number: EHR Cellulose hydrolysis to glucose is most often catalyzed using mineral acids or enzymes (cellulases). Cellulase hydrolysis is preferred over mineral acid hydrolysis for several reasons: acid hydrolysis leads to formation of undesirable degradation products of glucose that significantly lower glucose yield and inhibit subsequent fermentation (Larsson et al., 1999; Sreenath and Jeffries, 2000; Taherzadeh et al., 1999); requires expensive corrosionresistant materials; and poses disposal problems. Glucose degradation products observed with acid pretreatment or hydrolysis include hydroxymethyl furfural (HMF) and furfural. In presence of water, HMF produces levunic acid and formic acid (Mosier et al., 2002) which inhibit downstream fermentation to ethanol. On the other hand, cellulase enzymes are very specific in their action, producing virtually no glucose degradation products. Cellulases (of fungal or bacterial origin) are in fact a mixture of enzymes which act in concert and synergistically (Mosier et al., 1999). Special materials of construction are not required with cellulase-catalyzed hydrolysis. However, cellulose hydrolysis in aqueous media suffers from slow reaction rates because the substrate (cellulose) is a waterinsoluble crystalline biopolymer. Therefore, the enzymes have to accomplish the hydrolytic decomposition via first adsorbing on the cellulose surface, partially stripping the individual polymer chains from the crystal structure, and then cleaving the glycoside bonds in the chain (Lynd et al., 2002; Zhang and Lynd, 2004). Adsorption sites of crystalline cellulose are very limited due to the tight packing arrangement of cellulose fibrils which not only excludes the enzymes but also largely excludes water (Lynd et al., 2002; Zhang and Lynd, 2004). Pretreatment methods increase the surface area accessible to water and cellulases are expected to generate improvements in hydrolysis kinetics and conversion of cellulose to glucose (Gollapalli et al., 2002; Sun and Cheng, 2002; Zhang and Lynd, 2004). Cellulose is very difficult to dissolve due to the extensive network of inter- and intra-molecular hydrogen bonds and van der Waals interactions between cellulose fibrils. Until ß2006 Wiley Periodicals, Inc.

2 recently, the best dissolution results could be found using non-derivatizing solvents such as molten salts (i.e., LiCl3H 2 O) and non-aqueous solvent systems such as N,Ndimethylacetamide/LiCl, DMSO/SO 2 and N-methylmorpholine-N-oxide (Heinze and Liebert, 2001). Enhancement of hydrolysis kinetics with pretreated cellulose was seen in early studies by Ladisch et al. (1978) using cadoxen (a mixture of 5% cadmium oxide in 28% aqueous ethylene diamine). Cellulosic materials were softened by soaking in cadoxen for 12 h at room temperature, rinsed and subsequently saccharified using cellulase. Saccharification reaction rates were enhanced compared to untreated controls. The authors attributed the increased kinetics to disruption of the cellulose crystalline structure. The long incubation time, toxicity of the cadoxen solvent and difficulty in recovery and reuse of the diamine limit this pretreatment route. Similar results have been obtained by incubating cellulose first in a solution of an iron/sodium tartrate complex followed by addition of a concentrated sodium hydroxide solution (iron precipitates as a hydroxide) (Hamilton et al., 1984). More recently, phosphoric acid, at concentrations ranging between 70 and 81%, was shown to swell and dissolve cellulose at higher concentrations (Hudson and Cuculo, 1980; Wood, 1988). Zhang et al. (Zhang et al., 2006; Zhang and Lynd, 2005) used ice-cold concentrated phosphoric acid to dissolve cellulose followed by its precipitation by diluting the mixture with ice-cold water. Enzymatic hydrolysis rates were found to increase by one to two orders of magnitude. Cellulose swelling and dissolution with other concentrated acids (sulfuric acid, hydrochloric acid, and nitric acid) have also been reported (Hudson and Cuculo, 1980). Acid pretreatment methods normally require corrosion resistant materials of construction and some mineral acids produce degradation products (Mosier et al., 2001). Ionic liquids (ILs) show promise as efficient novel solvents for dissolution and pretreatment of cellulose. Their ability to dissolve large amounts of cellulose at considerably mild conditions and the feasibility of recovering 100% of the used IL to its initial purity makes them attractive (Heinze et al., 2005). Recently, solubility of up to 39, 25, and 10% (w/w) has been reported for the ILs 3-methyl-N-butylpyridinium chloride ([C 4 mpy]cl) (Heinze et al., 2005), 1-n-butyl-3- methylimidazolium chloride ([C 4 mim]cl) (Swatloski et al., 2002), and 1-allyl-3-methylimidazolium chloride (Wu et al., 2004), respectively. ILs are salts that are liquids near room temperature and are stable up to temperatures of about 3008C. With their low volatility, fluidity at ambient temperatures, and unique solvent properties, ILs comprise a class of prospective solvents that are potentially green due to their minimal air emissions. Their toxicity to animal and aquatic life has not been thoroughly evaluated to date. In our approach, [C 4 mim]cl, a non-derivatizing solvent (Moulthrop et al., 2005), was used to dissolve cellulose followed by rapid precipitation with an anti-solvent such as water or alcohol. The regenerated cellulose exhibited improved enzymatic hydrolysis kinetics. The structure of the regenerated cellulose was examined using X-ray powder diffraction (XRD) and found to lack the crystallinity of untreated cellulose. An important aspect of our approach is that the IL is able to instantly reject (precipitate) all the dissolved cellulose in presence of anti-solvents such as water, methanol, and ethanol via a preferential solute-displacement mechanism. Once the cellulose is precipitated, the anti-solvent used for displacement can easily be stripped off the non-volatile IL via flash distillation and the IL recovered for subsequent reuse. MATERIALS AND METHODS Reagents Microcrystalline cellulose, Avicel PH-101 (FMC Corp., Philadelphia, PA) and the glucose hexokinase assay reagents were obtained from Sigma Aldrich (St. Louis, MO). Citric acid monohydrate, sodium citrate, 3,5-dinitrosalicylic acid (DNS), sodium hydroxide, sodium potassium tartarate (rochelle salt), phenol, sodium metabisulfite, methanol, and ethanol were obtained from Fisher Scientific (Hanover Park, IL). 1-n-butyl-3-methylimidazolium chloride ([C 4 mim]cl) was purchased from Lancaster Synthesis (Alfa Aesar, Pelham, NH) and used without further purification. Cellulase, from Trichoderma reesei (ATCC#26799) was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Cellulase activity was determined by the standard filter paper assay and expressed as filter paper units per gram of glucan (FPU) (Ghose, 1987). One FPU is defined as the enzyme that releases 1 mmol of glucose equivalents per minute from Whatman No.1 filter paper. The released reducing sugars were measured by the DNS method using D-glucose as a standard (Miller, 1959). The rate of formation of these soluble reducing sugars (i.e., glucose, cellobiose, and higher soluble oligodextrins) is taken as a measure of the rate of cellulose hydrolysis. In addition, released glucose was also determined separately using a hexokinase/glucose-6-phosphate assay (Bondar and Mead, 1974). Cellulose Pretreatment and Regeneration A 5% (w/w) cellulose solution was prepared by combining 50 mg of cellulose with 950 mg [C 4 mim]cl in a 5 ml autoclave vial. All samples were prepared under a nitrogen atmosphere to prevent uptake of water by the IL. The vial and the contents were heated in a block heater to 130, 140, or 1508C and incubated for 10, 30, 60, 120, or 180 min. The samples were gently stirred by placing the block heater on an orbital shaker. Deionized water, methanol, and ethanol were used as antisolvents for precipitating cellulose from [C 4 mim]cl. About 2 ml of anti-solvent was added to the cellulose/[c 4 mim]cl mixture. A precipitate immediately formed. The sample was briefly centrifuged and supernatant was removed. The sample was washed five to six times with additions of antisolvent, centrifuged and supernatant removed. Buffer solution used in the cellulose hydrolysis step was used for the final Dadi et al.: Cellulose Saccharification Kinetics 905 Biotechnology and Bioengineering. DOI /bit

3 washes. The resultant cellulose is referred to as regenerated cellulose. Enzymatic Hydrolysis Batch enzymatic hydrolysis of regenerated and untreated cellulose was carried out at 508C in a reciprocating shaker bath. Total batch volume was approximately 3 ml with an enzyme concentration of FPU/g glucan, and substrate concentration of about 17 mg/ml. Solutions were buffered with 0.05 M sodium citrate, ph 4.8. The enzyme reaction was monitored by withdrawing samples from the supernatant periodically and measuring release of soluble reducing sugars by the DNS assay (Miller, 1959) and glucose using a hexokinase glucose assay (Bondar and Mead, 1974). Untreated and regenerated cellulose were hydrolyzed using the same cellulase (from T. reesei) stock solution. The untreated cellulose controls were run concurrently with all regenerated cellulose hydrolysis experiments to eliminate potential differences in temperature history or enzyme loading. XRD Measurements Smooth films were cast at room temperature with regenerated cellulose on microscope slides. XRD data for these films were generated at 258C with an X PERT PRO powder diffractometer PANalytical with X celerator detector using Nickel filtered CuKa radiation. Samples were scanned over the angular range ,2y, with a step size of 0.058, and step time of 10 s. RESULTS Hydrolysis and Glucose Formation Rates of Regenerated and Untreated Cellulose Avicel, a microcrystalline cellulose, was regenerated by dissolution in 1-n-butyl-3-methylimidazolium chloride ([C 4 mim]cl) followed by precipitation with an anti-solvent (water, methanol, or ethanol). Alternative anti-solvents methanol and ethanol were examined because [C 4 mim]cl is more easily recovered, following cellulose regeneration, from volatile organic solvents than water through a simple distillation step. The resulting hydrolysis rates for cellulose regenerated with water and the solvents as measured by total soluble reducing sugars (DNS) and conversion of cellulose to glucose are shown in Figure 1. The initial rates of hydrolysis of cellulose to soluble reducing sugars are shown in Table I. Initial rates of enzymatic hydrolysis of regenerated cellulose were at least 50 times that of untreated cellulose. Alcohol anti-solvents appeared to result in somewhat higher initial saccharification rates (Table I). Both the hydrolysis rates and rates of glucose formation were significantly greater for the regenerated cellulose samples compared to those for untreated cellulose (Fig. 1). Figure 1. Avicel samples were incubated for 10 min in [C 4 mim]cl at 1308C, and precipitated with either ethanol (&), deionized water (*), or methanol (~). Hydrolysis rates of IL-incubated samples are compared to that of untreated Avicel ("). Conversion of cellulose to sugars for batch samples of 17 mg/ml Avicel hydrolyzed with T. reesei cellulase activity of 60 FPU/g glucan at 508C is shown as a function of time for (a) total soluble sugars (measured using a DNS assay) and (b) as glucose (measured by glucose hexokinase assay). The amount of reducing sugars released from regenerated and untreated cellulose during the first 3 h of enzymatic hydrolysis reaction were approximately 13 and 2.6 mg/ml and the amount of glucose liberated for regenerated and untreated cellulose were approximately 6.5 and 2.3 mg/ml (Fig. 1). This implies that a net 6.5 mg/ml of soluble cellodextrins are formed for regenerated cellulose and 0.3 mg/ml for untreated cellulose in the first 3 h. Role of Incubation Time and Temperature The incubation time for samples dissolved in [C 4 mim]cl at 1308C and precipitated with water was varied from 10 min to 906 Biotechnology and Bioengineering, Vol. 95, No. 5, December 5, 2006 DOI /bit

4 Table I. Initial rate of formation of total soluble reducing sugars measured by DNS assay in enzymatic hydrolysis of Avicel cellulose. Anti-solvent Initial rate of formation of soluble reducing sugars (mg/ml/min) Rate enhancement a Water Methanol Ethanol Untreated Regenerated or untreated Avicel samples (17 mg/ml) were hydrolyzed using a cellulase activity of 60 FPU/g glucan. Regenerated cellulose samples were incubated for 10 min at 1308C and precipitated using the anti-solvents water, methanol, or ethanol. Rates are calculated from data obtained in the first 20 min of hydrolysis. a Rate enhancement is defined as the ratio of initial rate of reducing sugars released for regenerated cellulose divided by that of untreated cellulose. 3 h. The regenerated cellulose samples were then hydrolyzed in a batch system and conversion of cellulose to glucose was calculated from measurements of glucose concentration at a number of time intervals. From the hydrolysis reaction stoichiometry, 1 g of cellulose upon complete hydrolysis produces 1.11 g of glucose. As seen in Figure 2, incubation time appears to have little effect on rate of glucose formation. There are marked differences in the rates of glucose formation between untreated and regenerated cellulose. For example, after 12 h approximately 59% of the regenerated cellulose is converted to glucose compared to 30% of the untreated cellulose. The IL temperature was varied from 130 to 1508C for the cellulose dissolution step. After incubation in the IL for 2 h, cellulose was precipitated with water. As seen in Figure 3, the rate of glucose formation for the resultant regenerated Figure 2. Effect of incubation time on glucose production. Conversion of cellulose to glucose (measured by glucose hexokinase assay) as a function of time for batch samples of 15 mg/ml Avicel hydrolyzed with 50 FPU/g glucan T. reesei cellulase at 508C. Samples were dissolved then incubated in [C 4 mim]cl at 1308C for 10 min (&), 30 min (*), 1 h (~), or 3 h (!) and precipitated with deionized water. The rate of conversion of cellulose to glucose appears unaffected by incubation time. The rate of glucose formation of incubated samples are higher than that of untreated Avicel ("). Figure 3. Effect of dissolution temperature on hydrolysis kinetics. Conversion of cellulose to glucose (measured by glucose hexokinase assay) as a function of time for batch samples of 17 mg/ml Avicel hydrolyzed with 115 FPU/g glucan T. reesei cellulase at 508C. Samples were incubated for 2 h in [C 4 mim]cl at 1308C(&), 1408C(*), or 1508C(~), and precipitated with deionized water. Incubation temperature of the cellulose/[c 4 mim]cl mixture does not appear to affect the rate of conversion of cellulose to glucose. Rate of glucose formation of incubated samples are higher than that of untreated Avicel ("). cellulose appears to be largely unaffected by this variation in temperature, although cellulose conversion to glucose appears to be slightly lower for the 1508C samples. The rates of glucose formation for regenerated cellulose again were found to be greater than those for untreated cellulose. After 12 h approximately 72% of the regenerated cellulose is converted to glucose compared to 32% of the untreated cellulose. The enzyme concentration for the hydrolysis experiments shown in Figure 3 was about two times that used for data shown in Figure 2. Interestingly, the conversion of cellulose to glucose after 12 h of hydrolysis was relatively unchanged for untreated cellulose with the increased enzyme concentration (30 vs. 32%) whereas conversion increased from 59 to 72% for regenerated cellulose. These results imply that available enzyme adsorption sites are saturated in the case of untreated cellulose and regeneration of cellulose produces additional sites for enzyme adsorption. Since incubation time and temperature do not appear to affect the subsequent hydrolysis of regenerated cellulose to glucose it appears that these variables need only to be adjusted to achieve complete dissolution of cellulose in the IL in order to produce regenerated cellulose which is more tractable to hydrolysis. Structure of Regenerated Cellulose To gain insight into the possible mechanism for enhancement of hydrolysis kinetics, the structure of regenerated and untreated cellulose was examined by XRD. As seen in Figures 4 and 5 regenerated cellulose is essentially amorphous and untreated cellulose is highly crystalline. Dadi et al.: Cellulose Saccharification Kinetics 907 Biotechnology and Bioengineering. DOI /bit

5 Figure 4. X-ray powder diffraction patterns for regenerated and untreated Avicel. Untreated Avicel, (A), exhibited a significantly greater degree of crystallinity than that of regenerated samples (B and C). Regenerated samples were incubated in [C 4 mim]cl at 1308C for (B) 2 h or (C) 30 min and precipitated with deionized water. Incubation time and anti-solvent selection does not appear to affect the resulting regenerated cellulose structure as measured by XRD. XRD results suggest that during pretreatment with IL and anti-solvent cellulose crystallinity is disrupted. Rapid precipitation with anti-solvent may prevent the restructuring of the dissolved cellulose into its crystalline form. DISCUSSION The difference in the amount of total reducing sugars released for regenerated (13.0 mg/ml) and untreated Figure 5. X-ray powder diffraction patterns of untreated and regenerated Avicel. Untreated Avicel, (A), exhibited a significantly greater degree of crystallinity than that of regenerated samples (B) through (D). Regenerated samples were incubated in [C 4 mim]cl at 1308C for 2 h and precipitated with (B) deionized water, (C) methanol, or (D) ethanol. cellulose (2.6 mg/ml) during the first 3 h of enzymatic hydrolysis (Fig. 1a) can be attributed to the differences in the cellulose structure for the amorphous regenerated cellulose and largely crystalline untreated cellulose. After about 2 h of enzymatic hydrolysis, the regenerated cellulose reaction mixtures appeared transparent whereas untreated cellulose mixtures were opaque. Cellodextrins (b-1,4-glucose oligomers) with a degree of polymerization (DP) from 2 to 6 are soluble in water (Klemm et al., 1998; Pereira et al., 1988; Zhang and Lynd, 2005) and cellodextrins with DP from 7 to 13 are slightly soluble in hot water (Schmid et al., 1988; Zhang and Lynd, 2003, 2004). Hence, the regenerated cellulose substrate appears to be rapidly hydrolyzed to short glucose oligomers, mitigating mass transfer limitations on overall reaction rates. Regenerated cellulose is hypothesized to have a higher fraction of b-glucosidic bonds accessible to cellulase due to the decreased crystallinity and potentially increased surface area obtained from the pretreatment process. Endoglucanases act at random sites in the internal accessible regions of cellulose polysaccharide chains, resulting in a rapid decrease in DP of the substrate due to the formation of smaller cellulose chains of varying lengths (Zhang and Lynd, 2004). Exoglucanase or cellobiohydrolase acts on the chain ends liberated by the action of endoglucanase, producing primarily cellobiose (Lynd et al., 2002; Teeri, 1997). In native cellulose, the low surface area limits the endoglucanases accessibility to b-glucosidase bonds and hence the decrease in DP is not as rapid as in the regenerated cellulose and the native cellulose remains insoluble. The early conversion of regenerated cellulose to soluble oligomers may allow easy isolation and separation of the cellulase enzymes from products and substrate, with recovery and recycle of enzyme (Gregg and Saddler, 1996; Lu et al., 2002; Steele et al., 2005). Steele et al. recovered high fractions of cellulase and cellobiase following hydrolysis of ammonia fiber explosion treated corn stover. As costs of enzymes remain a limiting factor in the manufacture of ethanol from cellulosic biomass, recovery of enzymes can account for considerable reductions in costs (Steele et al., 2005). The high rate of conversion of cellulose to soluble cellodextrins may allow the use of lower cellulase loadings with the supplement of b-glucosidase, which will also reduce the costs of enzymes. Dissolution of cellulose in the IL and its subsequent precipitation with anti-solvent, allows separation of the IL/ solvent mixture from cellulose by a simple filtration or centrifugation step. The IL and anti-solvent can then be recovered, easily separated and recycled. The dissolution mechanism of cellulose in [C 4 mim]cl can be attributed to the nature of the bulky imidazolium cation and the relatively strong electronegativity and small size of the chloride ion. [C 4 mim]cl has high hydrogen bond basicity and the anion plays a key role in the dissolution of cellulose. The chloride ion attacks the free hydroxyl groups and deprotonates cellulose. The imidazolium cation with its electron rich aromatic p system interacts with cellulose hydroxyl oxygen atoms via non-bonding or p electrons and in addition 908 Biotechnology and Bioengineering, Vol. 95, No. 5, December 5, 2006 DOI /bit

6 prevents in the crosslinking of the cellulose molecules (Anderson et al., 2002; Zhang et al., 2005). During precipitation with anti solvents, [C 4 mim]cl is extracted into the anti-solvent because of interactions of IL and anti solvent due to hydrogen bonding, dipolar, and coulombic forces with solvent (Crosthwaite et al., 2005). CONCLUSIONS In our approach, [C 4 mim]cl was used to dissolve cellulose followed by rapid precipitation with an anti-solvent such as water or alcohol. The resultant regenerated cellulose was amorphous in structure allowing a greater number of sites for enzyme adsorption with a subsequent enhancement of hydrolysis kinetics. The regenerated cellulose exhibited improved hydrolysis kinetics with optically transparent solutions formed after about 2 h of reaction. This may provide an opportunity for separation of products and unreacted substrate from the catalyst (enzyme) easing enzyme recovery. Due to the non-volatility of the IL, antisolvent can be easily stripped from the IL/solvent mixture for recovery and recycle of both the IL and anti-solvent. The authors would like to thank Dr. Sudhir Aki and Dr. Joan Brennecke of the University of Notre Dame, Department of Chemical and Biomolecular Engineering, for helpful discussions and ionic liquid samples. References Anderson JL, Ding J, Welton T, Armstrong DW Characterizing ionic liquids on the basis of multiple solvation interactions. J Am Chem Soc 124: Bondar RJ, Mead DC Evaluation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides in the hexokinase method for determining glucose in serum. Clin Chem 20(5): Crosthwaite JM, Aki SNVK, Maginn EJ, Brennecke JF Liquid phase behavior of imidazolium-based ionic liquids with alcohols: Effect of hydrogen bonding and non-polar interactions. Fluid Phase Eq : Ghose TK Measurement of cellulase activities. Pure Appl Chem 59(2): Gollapalli LE, Dale BE, Rivers DM Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw. Appl Biochem Biotechnol 98(100): Gregg DJ, Saddler JN Factors affecting cellulose hydrolysis and the poteintial of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnol Bioeng 51: Hamilton TJ, Dale BE, Ladisch MR, Tsao GT Effect of ferric tartrate/sodium hydroxide solvent pretreatment on enzyme hydrolysis of cellulose in corn residue. Biotechnol and Bioeng 26: Heinze T, Liebert T Unconventional methods in cellulose functionalization. Prog Polymer Sci 26: Heinze T, Schwikal K, Barthel S Ionic liquids as reaction medium in cellulose functionalization. Macromol Biosci 5: Hudson SM, Cuculo AJ The solubility of unmodified cellulose: A critique of the literature. J Macromol Sci Rev Macromol Chem C18(1):1 82. Klemm D, Philipp B, Heinze T, Heinze U, Wagenknecht W Comprehensive cellulose chemistry. I. Fundamentals and analytical methods. Weinheim: Wiley-VCH. Ladisch MR, Ladisch CM, Tsao GT Cellulose to sugars: New path gives quantitative yield. Science 201: Larsson S, Palmqvist E, Hahn-hagerdal B, Tengborg C, Stenberg K, Zacchi G, Nilvebrant N The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol 24: Lu Y, Yang B, Gregg D, Saddler JN, Mansfield SD Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam exploded softwood residues. Appl Biochem Biotechnol 98(100): Lynd LR, Wyman CE, Gerngross TU Biocommodity engineering. Biotechnol Prog 15: Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3): Mansfield SD, Mooney C, Saddler JN substrates and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Prog 15(5): Miller GL Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3): Mosier NS, Hall P, Ladisch CM, Ladisch MR Reaction kinetics, molecular action, and mechanism of cellulolytic proteins. Adv Biochem Eng/Biotechnol 65:23. Mosier NS, Sarikaya A, Ladisch CM, Ladisch MR Characterization of dicarboxylic acids for cellulose hydrolysis. Biotechnology Prog 17: Mosier NS, Ladisch CM, Ladisch MR Characterization of acid catalytic domains for cellulose hydrolysis and glucose degradation. Biotechnol Bioeng 79(6): Moulthrop JS, Swatloski RP, Moyna G, Rogers RD High resoution 13C-NMR studies of cellulose and cellulose oligimers in ionic liquid solutions. Chem Comm 12: Pereira AN, Mobedshahni M, Ladisch MR Preparation of cellodextrins. Methods Enzymol 160: Pizzi A, Eaton N The structure of cellulose by confirmational analysis 2. The cellulose polymer chain. J Macromol Sci Chem 22: Schmid G, Bisell M, Wandrey C Preparation of cellodextrins and isolation of oligomeric side components and their characterization. Anal Biochem 175(2): Sreenath HK, Jeffries TW Production of ethanol from wood hydrolyzate by yeasts. Bioresource Tech 72: Steele B, Raj S, Nghiem J, Stowers M Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion treated corn stover. Appl Biochem Biotechnol 124: Sun Y, Cheng J Hydrolysis of lignocellulosic materials for ethanol production: A review. Biosource Tech 83:1 11. Swatloski RP, Spear SK, Holbrey JD, Rogers RD Dissolution of cellulose with ionic liquids. J Am Chem Soc 124: Taherzadeh MJ, Niklasson C, Liden G Conversion of dilute acid hydrolyzates of spruce and birch to ethanol by fed batch fermentation. Bioresource Tech 69: Teeri TT Crystalline cellulose degradation: New insight into the function of cellobiohydrolases. Trends Biotechnol 15: Wood TM Preparation of crystalline, amorphous, and dyed cellulase substrates. Methods Enzymol 160: Wu J, Zhang J, He J, Ren Q, Guo M Homogeneous acetylation of cellulose in a new ionic liquid. Biomacromolecules 5:266. Zhang Y-HP, Lynd LR Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation. Anal Biochem 322: Dadi et al.: Cellulose Saccharification Kinetics 909 Biotechnology and Bioengineering. DOI /bit

7 Zhang Y-HP, Lynd LR Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnol Bioeng 88(7): Zhang Y-HP, Lynd LR Determination of the numberaverage degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis. Biomacromolecules 6: Zhang H, Wu J, Zhang J, He J Allyl-3-methylimidazolium chloride room temperature ionic liquid: A new and powerful nonderivatizing solvent for cellulose. Macromolecules 38(20): Zhang Y-HP, Cui J, Lynd LR, Kuang LR A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7(2): Biotechnology and Bioengineering, Vol. 95, No. 5, December 5, 2006 DOI /bit