Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments

Attempts were made to enhance cellulose saccharification by cellulase using cellulose dissolution as a pretreatment step. Four cellulose dissolution agents, NaOH/Urea solution, N-methylmorpholine-N-oxide (NMMO), ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and 85% phosphoric acid were employed to dissolve cotton cellulose. In comparison with conventional cellulose pretreatment processes, the dissolution pretreatments were operated under a milder condition with temperature <130 °C and ambient pressure. The dissolved cellulose was easily regenerated in water. The regenerated celluloses exhibited a significant improvement (about 2.7- to 4.6-fold enhancement) on saccharification rate during 1st h reaction. After 72 h, the saccharification yield ranged from 87% to 96% for the regenerated celluloses while only around 23% could be achieved for the untreated cellulose. Even with high crystallinity, cellulose regenerated from phosphoric acid dissolution achieved the highest saccharification rates and yield probably due to its highest specific surface area and lowest degree of polymerization (DP).     

Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac])

In this study, sugarcane bagasse was pretreated by six ionic liquids (ILs) using a bagasse/IL ratio of 1:20 (wt%). The solubilization of bagasse in the ILs was followed by water precipitation. On using 1-ethyl-3-methylimidazolium acetate [Emim] [Ac] at 120 °C for 120 min, 20.7% of the bagasse components remained dissolved and enzymatic saccharification experiments resulted on 80% glucose yield within 6h, which evolved to over 90% within 24 h. Moreover, FE-SEM analysis of the precipitated material indicated a drastic lignin extraction and the exposure of nanoscopic cellulose microfibrils with widths of less than 100 nm. The specific surface area (SSA) of the pretreated bagasse (131.84 m2/g) was found to be 100 times that of untreated bagasse. The ability of [Emim] [Ac] to simultaneously increase the SSA and to decrease the biomass crystallinity is responsible for the improved bagasse enzymatic saccharification rates and yields obtained in this work.     

Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis

Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass (Panicum virgatum), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate ([C2mim][OAc]) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin-carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.     

Sugarcane bagasse pretreatment using three imidazolium-based ionic liquids; mass balances and enzyme kinetics

ABSTRACT: BACKGROUND: Effective pretreatment is key to achieving high enzymatic saccharification efficiency in processing lignocellulosic biomass to fermentable sugars, biofuels and value-added products. Ionic liquids (ILs), still relatively new class of solvents, are attractive for biomass pretreatment because some demonstrate the rare ability to dissolve all components of lignocellulosic biomass including highly ordered (crystalline) cellulose. In the present study, three ILs, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc) are used to dissolve/pretreat and fractionate sugarcane bagasse. In these IL-based pretreatments the biomass is completely or partially dissolved in ILs at temperatures greater than 130[DEGREE SIGN]C and then precipitated by the addition of an antisolvent to the IL biomass mixture. For the first time mass balances of IL-based pretreatments are reported. Such mass balances, along with kinetics data, can be used in process modelling and design. RESULTS: Lignin removals of 10% mass of lignin in bagasse with [C4mim]Cl, 50% mass with [C2mim]Cl and 60% mass with [C2mim]OAc, are achieved by limiting the amount of water added as antisolvent to 0.5 water:IL mass ratio thus minimising lignin precipitation. Enzyme saccharification (24 h, 15FPU) yields (% cellulose mass in starting bagasse) from the recovered solids rank as: [C2mim]OAc(83%)>[C2mim]Cl(53%) = [C4mim]Cl(53%). Composition of [C2mim]OAc-treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are characteristic of aqueous alkali pretreatments while those of chloride IL-treated solids resemble aqueous acid pretreatments. All ILs are fully recovered after use (100% mass as determined by ion chromatography). CONCLUSIONS: In all three ILs regulated addition of water as an antisolvent effected a polysaccharide enriched precipitate since some of the lignin remained dissolved in the aqueous IL solution. Of the three IL studied [C2mim]OAc gave the best saccharification yield, material recovery and delignification. The effects of [C2mim]OAc pretreatment resemble those of aqueous alkali pretreatments while those of [C2mim]Cl and [C4mim]Cl resemble aqueous acid pretreatments. The use of imidazolium IL solvents with shorter alkyl chains results in accelerated dissolution, pretreatment and degradation.     

Biological pretreatment of cellulose: enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases

In this study, cellulose-binding domains (CBDs) of cellulases from Trichoderma reesei were used in a pretreatment step and were found to effectively reduce the crystallinity of cellulose (both Avicel and fibrous cellulose). This, in turn, led to higher glucose concentrations (up to 25% increase) in subsequent hydrolysis of cellulose using a mixture of cellulases and without the need for any intermediate purification step. CBDs were shown to be active in a range of temperatures (up to 50°C), while cellulase hydrolytic activity was greatly reduced after incubation at 50°C. This was explained by retention of full binding capacity after incubation at 50°C for 15 h. Our findings suggest that CBDs may be a valuable tool in pretreating cellulose and eventually afford faster enzymatic conversion of cellulose to glucose, thus contributing to more affordable processes in the production of biofuels.     

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.     

Dissolution of cellulose in ionic liquid and water mixtures as revealed by molecular dynamics simulations

Abstract

Increasing population growth and industrialization are continuously oppressing the existing energy resources, elevating the pollution and global fuel demand. Various alternate energy resources can be utilized to cope with these problems in an environment-friendly fashion. Currently, bioethanol (sugarcane, corn-derived) is one of the most widely consumed biofuels in the world. Lignocellulosic biomass is yet another attractive resource for sustainable bioethanol production. Pretreatment step plays a crucial role in the lignocellulose to bioethanol conversion by enhancing cellulose susceptibility to enzymatic hydrolysis. However, economical lignocellulose pretreatment still remains a challenging job. Ionic liquids (ILs), especially 1-ethyl-3-methylimidazolium acetate (EmimAc), is an efficient solvent for cellulose dissolution with improved enzymatic saccharification kinetics. To increase the process efficiency as well as recyclability of IL, water is shown as a compatible cosolvent for lignocellulosic pretreatment. The performance analysis of IL–water mixture based on the molecular level understanding may help to design effective pretreatment solvents. In this study, all-atom molecular dynamics simulation has been performed using EmimAc–water mixtures to understand the behavior of cellulose microcrystal containing eight glucose octamers at room and pretreatment temperatures. High-temperature simulation results show effective cellulose chain separation where cellulose–acetate interaction is found to be the driving force behind dissolution. It is also observed that pretreatment with 50 and 80% IL mixture is efficient in decreasing cellulose crystallinity. At a high IL concentration, water exists in a clustered network which gradually spans into the medium with increasing water fraction leading to loss of its cosolvation activity.

Communicated by Ramaswamy H. Sarma     

Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by N-methylmorpholine-N-oxide

Pretreatment of lignocellulosic biomass by N-methylmorpholine-N-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity after dissolution and precipitation processes. This work focused on assessing several physical changes and their correlations to enzymatic saccharification of pine flour after NMMO pretreatment. Results from microstructure, surface chemical composition, and cellulose accessibility complementarily illustrated the enrichment of cellulose on pine flour surface after NMMO pretreatment. Cellulose accessibility was highly correlated to the overall glucan conversion rate. Changes in crystallinity were correlated to the initial hydrolysis rate but not overall glucan conversion rate. Findings from this work may contribute to lignocellulosic bioenergy from development of novel pretreatment technologies utilizing NMMO.     

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus

ABSTRACT: BACKGROUND: Lignocellulose is the most abundant biomass on earth. However, biomass recalcitrance has become a major factor affecting biofuel production. Although cellulose crystallinity significantly influences biomass saccharification, little is known about the impact of three major wall polymers on cellulose crystallization. In this study, we selected six typical pairs of Miscanthus samples that presented different cell wall compositions, and then compared their cellulose crystallinity and biomass digestibility after various chemical pretreatments. RESULTS: A Miscanthus sample with a high hemicelluloses level was determined to have a relatively low cellulose crystallinity index (CrI) and enhanced biomass digestibility at similar rates after pretreatments of NaOH and H2SO4 with three concentrations. By contrast, a Miscanthus sample with a high cellulose or lignin level showed increased CrI and low biomass saccharification, particularly after H2SO4 pretreatment. Correlation analysis revealed that the cellulose CrI negatively affected biomass digestion. Increased hemicelluloses level by 25% or decreased cellulose and lignin contents by 31% and 37% were also found to result in increased hexose yields by 1.3-times to 2.2-times released from enzymatic hydrolysis after NaOH or H2SO4 pretreatments. The findings indicated that hemicelluloses were the dominant and positive factor, whereas cellulose and lignin had synergistic and negative effects on biomass digestibility. CONCLUSIONS: Using six pairs of Miscanthus samples with different cell wall compositions, hemicelluloses were revealed to be the dominant factor that positively determined biomass digestibility after pretreatments with NaOH or H2SO4 by negatively affecting cellulose crystallinity. The results suggested potential approaches to the genetic modifications of bioenergy crops.     

Effects of sediment pretreatment on the performance of sediment microbial fuel cells

In the present study, the effects of different pretreatment methods for sediments on the performance of sediment microbial fuel cells (SMFCs) were evaluated. Autoclaved (30 and 60 min), and heated (150 °C, 3 h) sediments demonstrated high power density, compared with control and heated (60 °C, 3 h) sediments. An SMFC with heated (60 °C, 3 h) sediment was found to easily form a biocathode. The power density of an SMFC with heated (150 °C, 3 h) sediment was 214 mW m(-2) on day 24. Furthermore, autoclaved (30 and 60 min) and heated (3 h, 60 and 150 °C) sediments accelerated the production of dissolved organic matter (DOM). The DOM in heated (60 °C, 3 h) sediments had larger molecular sizes. The present study demonstrates that SMFCs can have high power density and high loss on ignition removal efficiencies when produced from sediments by suitable pretreatment methods.     

Short time ionic liquids pretreatment on lignocellulosic biomass to enhance enzymatic saccharification

The potential of 1-buthyl-3-methylpyridinium chloride, [Bmpy][Cl], as a pretreatment solvent for lignocellulosic biomasses, Bagasse and Eucalyptus, was investigated. The yields of regenerated biomasses ranged between 35% and 96%, and varied according to the pretreatment time, type of ionic liquid (IL) and biomass. The pretreatment of the biomass with [Bmpy][Cl] resulted in up to 8-fold increase in the cellulose conversion when compared with the untreated biomass. For a short pretreatment period (i.e., 10 min), [Bmpy][Cl] showed better performance than 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) with respect to the initial enzymatic saccharification rates. The increase in the reaction rates with [Emim][OAc] treatment was because of a reduction in the cellulose crystallinity. In contrast, a decrease in the crystallinity index was not clearly observed for the biomass pretreated with [Bmpy][Cl], and the enhancement of the enzymatic saccharification rates using this IL is presumably due to a reduction in the degree of polymerization of cellulose in the biomass.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

Structural comparison and enhanced enzymatic hydrolysis of the cellulosic preparation from Populus tomentosa Carr., by different cellulose-soluble solvent systems

This study aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharification. LiOH/urea, LiCl/DMAc, concentrated phosphoric acid, ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and N-methyl-morpholine-N-oxide (NMMO) were selected as the cellulose dissolution agents. Except the cellulosic sample regenerated from LiCl/DMAc system, all the other treated samples exhibited lower cellulose crystallinity and degree of polymerization (DP), and consequently, exhibited a significant enhancement on enzymatic hydrolysis kinetic. Ionic liquid pretreatment offered unique advantages in the hydrolysis rate in the first 10 h, probably due to the extensively structural transformation of cellulose from the crystalline to the amorphous region. Meanwhile, the regenerated cellulose from concentrated phosphoric acid almost completely consisted of cellulose II, and achieved the highest saccharification yield.     

Enhanced enzymatic saccharification of kenaf powder after ultrasonic pretreatment in ionic liquids at room temperature

This study demonstrates for the first time that the enzymatic hydrolysis of cellulose is drastically enhanced following ultrasonic pretreatment of lignocellulosic material in ionic liquids (ILs) when compared to conventional thermal pretreatment. Five types of ILs, 1-buthyl-3-methylimidazolium chloride (BmimCl), 1-allyl-3-methylimidazolium chloride (AmimCl), 1-ethyl-3-methylimidazolium chloride (EmimCl), 1-ethyl-3-methylimidazolium diethyl phosphate (EmimDep), and 1-ethyl-3-methylimidazolium acetate (EmimOAc) were tested. Cellulose saccharification ratio was about 20% for kenaf powders pretreated in BmimCl, AmimCl, EmimCl, and EmimDep by conventional heating at 110 °C for 120 min. Conversely, 60-95% of cellulose was hydrolyzed to glucose, subsequent to ultrasonic pretreatment in the same ILs for 120 min at 25 °C. The cellulose saccharification ratio of kenaf powder in EmimOAc was 86% after only 15 min of the ultrasonic pretreatment at 25 °C, compared to only 47% in that case of thermal pretreatment in the IL.     

Delignification of sugarcane bagasse using glycerol-water mixtures to produce pulps for saccharification

This paper describes the organosolv delignification of depithed bagasse using glycerol–water mixtures without a catalyst. The experiments were performed using two separate experimental designs. In the first experiment, two temperatures (150 and 190 °C), two time periods (60 and 240 min) and two glycerol contents (20% and 80%, v/v) were used. In the second experiment, which was a central composite design, the glycerol content was maintained at 80%, and a range of temperatures (141.7–198.3 °C) and time (23–277 min) was used. The best result, obtained with a glycerol content of 80%, a reaction time of 150 min and a temperature of 198.3 °C, produced pulps with 54.4% pulp yield, 7.75% residual lignin, 81.4% delignification and 13.7% polyose content. The results showed that high contents of glycerol tend to produce pulps with higher delignification and higher polyoses content in relation to the pulps obtained from low glycerol content reactions. In addition, the proposed method shows potential as a pretreatment for cellulose saccharification.     

Effects of preheating on briquetting and subsequent hydrothermal pretreatment for enzymatic saccharification of wheat straw

Briquetting of plant biomass with low bulk density is an advantage for handling, transport, and storage of the material, and heating of the biomass prior to the briquetting facilitates the densification process and improves the physical properties of the briquettes. This study investigates the effects of preheating prior to briquetting of wheat straw (WS) on subsequent hydrothermal pretreatment and enzymatic conversion to fermentable sugars. WS (11% moisture content) was densified to briquettes under different conditions; without preheating or with preheating at 75 or 125°C for either 5 or 10 min. Subsequent hydrothermal pretreatment was done for both un-briquetted WS and for briquettes. Enzymatic saccharification was afterwards performed for all samples. The results showed that as expected, nonpretreated WS briquettes gave very low sugar yields (22–29% of the cellulose content), even though preheating at 125°C prior to briquetting (without pretreatment) improved sugar yields somewhat. When combined with pretreatment, briquetting with preheating showed neutral or negative effects on sugar yield. This result suggests that moderate preheating (75°C for 5 min) before briquetting improved bulk density and compressive resistance of briquettes without impeding subsequent enzymatic conversion. However, excessive preheating (75 or 125°C for 10 min) before briquetting may result in irreversible structural modifications that hinder the interaction between biomass and water during pretreatment, thereby decreasing the accessibility of cellulose to enzymatic saccharification.     

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Wet oxidation pretreatment, enzymatic hydrolysis and simultaneous saccharification and fermentation of clover-ryegrass mixtures

The potential of clover (Trifolium repens) and ryegrass (Lolium perenne) mixtures as raw materials for ethanol production was investigated. Wet oxidation, at 175, 185 or 195 degrees C during 10min at two different oxygen pressures and with either addition or no addition of sodium carbonate, was evaluated as pretreatment method for clover-ryegrass mixtures. The enzymatic hydrolysis of cellulose was significantly improved after pretreatment. The highest conversion efficiency, 93.6%, was achieved for the sample pretreated at 195 degrees C, 10min, 1.2MPa and no addition of Na(2)CO(3). For that sample, the overall glucose yield after pretreatment and hydrolysis was 75.5%. No inhibition of cellulose enzymatic conversion by the filtrates was observed. The simultaneous saccharification and fermentation of the pretreated material yielded cellulose conversions of 87.5 and 86.6%, respectively, with Saccharomyces cerevisiae and the filamentous fungus Mucor indicus, and revealed that no addition of nutrients is needed for the fermentation of clover-ryegrass hydrolysates.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.