The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations.     

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.     

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose

Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose.     

Analysis of retted and non retted flax fibres by chemical and enzymatic means

Flax fibres (Linum usitatissimum L.) were subjected to chemical and enzymatic analysis in order to determine the compositional changes brought about by the retting process and also to determine the accessibility of the fibre polymers to enzymatic treatment. Chemical analysis involved subjecting both retted and non retted fibres to a series of sequential chemical extractions with 1% ammonium oxalate, 0.05 M KOH, 1 M KOH and 4 M KOH. Retting was shown to cause minimal weight loss from the fibres but caused significant changes to the pectic polymers present. Retted fibres were shown to have significantly lower amounts of rhamnogalacturonan as well as arabinan and xylan. In addition the average molecular mass of the pectic extracts was considerably lowered. Enzyme treatment of the 1 M KOH extracts with two different enzymes demonstrated that the non retted extract contained a relatively high molecular weight xylan not found in the retted extract. Treatment of the 1 M KOH extracts and the fibres with Endoglucanase V from Trichoderma viride demonstrated that while this enzyme solubilised cellulose as well as xylan and xyloglucan oligomers from the extract, it had limited access to these polymers on the fibre. MALDI-TOF MS analysis of the material solubilised from the extract suggested that the xylan was randomly substituted with 4-O-methyl glucuronic acid moieties. The xyloglucan was shown to be of the XXXG type and was substituted with galactose and fucose units. The enzyme treatments of the fibres demonstrated that the xylan and xyloglucan polymers in the fibres were not accessible to the enzyme but that material which was entrapped by the cellulose could be released by the hydrolysis of this cellulose.     

Enzymatic surface modification of cellulose acetate fibre by cutinase-CBM (carbohydrate-binding module) fusion proteins

Abstract

In this paper, two types of bacterial fusion protein, cutinase-CBM_Cel6A and cutinase-CBM_CenA, were used to modify the surface of cellulose acetate fibre. The enzyme binding on cellulose acetate fibres and the hydrolysis of acetyl groups were monitored. Water absorbency and dye uptake were measured to assess the extent of enzymatic modification. The results demonstrated that cutinase-carbohydrate-binding module (CBM) has a greater effect on cellulose acetate fibres than that of cutinase. The use of non-ionic surfactant Triton X-100 could further improve enzymatic modification of cellulose acetate fibres in terms of wettability and dyeability. Scanning electron microscopy confirmed that both cutinase-CBMs could lead to the formation of carving characters on the surface of treated cellulose acetate fibres. Our studies provide a foundation for the potential application of cutinase-CBM in the surface modification of cellulose acetate fibre.     

Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels

Toward exploiting the attractive mechanical properties of cellulose I nanoelements, a novel route is demonstrated, which combines enzymatic hydrolysis and mechanical shearing. Previously, an aggressive acid hydrolysis and sonication of cellulose I containing fibers was shown to lead to a network of weakly hydrogen-bonded rodlike cellulose elements typically with a low aspect ratio. On the other hand, high mechanical shearing resulted in longer and entangled nanoscale cellulose elements leading to stronger networks and gels. Nevertheless, a widespread use of the latter concept has been hindered because of lack of feasible methods of preparation, suggesting a combination of mild hydrolysis and shearing to disintegrate cellulose I containing fibers into high aspect ratio cellulose I nanoscale elements. In this work, mild enzymatic hydrolysis has been introduced and combined with mechanical shearing and a high-pressure homogenization, leading to a controlled fibrillation down to nanoscale and a network of long and highly entangled cellulose I elements. The resulting strong aqueous gels exhibit more than 5 orders of magnitude tunable storage modulus G' upon changing the concentration. Cryotransmission electron microscopy, atomic force microscopy, and cross-polarization/magic-angle spinning (CP/MAS) 13C NMR suggest that the cellulose I structural elements obtained are dominated by two fractions, one with lateral dimension of 5-6 nm and one with lateral dimensions of about 10-20 nm. The thicker diameter regions may act as the junction zones for the networks. The resulting material will herein be referred to as MFC (microfibrillated cellulose). Dynamical rheology showed that the aqueous suspensions behaved as gels in the whole investigated concentration range 0.125-5.9% w/w, G' ranging from 1.5 Pa to 105 Pa. The maximum G' was high, about 2 orders of magnitude larger than typically observed for the corresponding nonentangled low aspect ratio cellulose I gels, and G' scales with concentration with the power of approximately three. The described preparation method of MFC allows control over the final properties that opens novel applications in materials science, for example, as reinforcement in composites and as templates for surface modification.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose

The rates and extents of enzymatic cellulose hydrolysis of dilute acid pretreated corn stover (PCS) decline with increasing slurry concentration. However, mass transfer limitations are not apparent until insoluble solids concentrations approach 20% w/w, indicating that inhibition of enzyme hydrolysis at lower solids concentrations is primarily due to soluble components. Consequently, the inhibitory effects of pH-adjusted pretreatment liquor on the enzymatic hydrolysis of PCS were investigated. A response surface methodology (RSM) was applied to empirically model how hydrolysis performance varied as a function of enzyme loading (12-40mg protein/g cellulose) and insoluble solids concentration (5-13%) in full-slurry hydrolyzates. Factorial design and analysis of variance (ANOVA) were also used to assess the contribution of the major classes of soluble components (acetic acid, phenolics, furans, sugars) to total inhibition. High sugar concentrations (130g/L total initial background sugars) were shown to be the primary cause of performance inhibition, with acetic acid (15g/L) only slightly inhibiting enzymatic hydrolysis and phenolic compounds (9g/L total including vanillin, syringaldehyde, and 4-hydroxycinnamic acid) and furans (8g/L total of furfural and hydroxymethylfurfural, HMF) with only a minor effect on reaction kinetics. It was also demonstrated that this enzyme inhibition in high-solids PCS slurries can be approximated using a synthetic hydrolyzate composed of pure sugars supplemented with a mixture of acetic acid, furans, and phenolic compounds, which indicates that generally all of the reaction rate-determining soluble compounds for this system can be approximated synthetically.     

Real‐time detection of the morphological change in cellulose by a nanomechanical sensor

Up to now, experimental limitations have prevented researchers from achieving the molecular‐level understanding for the initial steps of the enzymatic hydrolysis of cellulose, where cellulase breaks down the crystal structure on the surface region of cellulose and exposes cellulose chains for the subsequent hydrolysis by cellulase. Because one of these non‐hydrolytic enzymatic steps could be the rate‐limiting step for the entire enzymatic hydrolysis of crystalline cellulose by cellulase, being able to analyze and understand these steps is instrumental in uncovering novel leads for improving the efficiency of cellulase. In this communication, we report an innovative application of the microcantilever technique for a real‐time assessment of the morphological change of cellulose induced by a treatment of sodium chloride. This sensitive nanomechanical approach to define changes in surface structure of cellulose has the potential to permit a real‐time assessment of the effect of the non‐hydrolytic activities of cellulase on cellulose and thereby to provide a comprehensive understanding of the initial steps of the enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2010;107: 190–194. © 2010 Wiley Periodicals, Inc.     

Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.     

Simultaneous saccharification and fermentation of cellulose to lactic acid

Recent interest in the industrial manufacture of ethanol and other organic chemicals from biomass has led to the utilization of surplus grain and cane juice as a fermentation feedstock. Since those starting materials are also foods, they are expensive. As an alternative, cellulosic substances-the most abundant renewable resources on earth(1)-have long been considered for conversion to readily utilizable hydrolyzates.(2, 3)For the production of ethanol from cellulose, we have proposed the simultaneous saccharification and fermentation (SSF) process.(4) In SSF, enzymatic cellulose hydrolysis and glucose fermentation to ethanol by yeast proceed simultaneously within one vessel. The process advantages-reduced reactor volume and faster saccharification rates-have been confirmed by many researchers.(5-8) During SSF, the faster saccharification rates result because the glucose product is immediately removed, considerably diminishing its inhibitory effect on the cellulase system.(9)To effectively apply the SSF method to produce substances fermented from glucose, several conditions should be satisfied. One is coincident enzymatic hydrolysis and fermentation conditions, such as pH and temperature. The other is that cellulase inhibition by the final product is less than that by glucose and/or cellobiose. One of us has reported that acetic acid, citric acid, itaconic acid, alpha-ketoglutaric acid, lactic acid, and succinic acid scarcely inhibit cellulase.(10) This suggests that if the microorganisms which produce these organic acids were compatible with cellulase reaction conditions, the organic acids could be produced efficiently from cellulosic substrates by SSF.In this article, the successful application of SSF to lactic acid production from cellulose is reported. Though there have been several reports of direct cellulose conversion to organic acids by anaerobes such as Clostridium, only trace amounts of lactic acid were detected in the fermentation medium among the low-molecular-weight fatty acid components.(11-13) Lactic acid is one of the most important organic acids and has a wide range of food-related and industrial applications.     

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

Background

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates.

Results

Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 °C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability, and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl₃-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose.

Conclusions

Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.

     

A pore‐hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass

Until now, most efforts to improve monosaccharide production from biomass through pretreatment and enzymatic hydrolysis have used empirical optimization rather than employing a rational design process guided by a theory‐based modeling framework. For such an approach to be successful a modeling framework that captures the key mechanisms governing the relationship between pretreatment and enzymatic hydrolysis must be developed. In this study, we propose a pore‐hindered diffusion and kinetic model for enzymatic hydrolysis of biomass. When compared to data available in the literature, this model accurately predicts the well‐known dependence of initial cellulose hydrolysis rates on surface area available to a cellulase‐size molecule. Modeling results suggest that, for particles smaller than 5 × 10^?3 cm, a key rate‐limiting step is the exposure of previously unexposed cellulose occurring after cellulose on the surface has hydrolyzed, rather than binding or diffusion. However, for larger particles, according to the model, diffusion plays a more significant role. Therefore, the proposed model can be used to design experiments that produce results that are either affected or unaffected by diffusion. Finally, by using pore size distribution data to predict the biomass fraction that is accessible to degradation, this model can be used to predict cellulose hydrolysis with time using only pore size distribution and initial composition data. Biotechnol. Bioeng. 2013; 110: 127–136. © 2012 Wiley Periodicals, Inc.     

Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.     

Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose

Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover.     

氨基磺酸应用于甘蔗渣预处理的研究

本研究尝试将氨基磺酸应用于甘蔗渣预处理,探究其作为酸预处理试剂对甘蔗渣成分和酶解的影响。氨基磺酸预处理最优条件为浓度3%,温度121℃,预处理1 h。在该条件下,甘蔗渣的固体回收率为64.45%,半纤维素和木质素去除率分别为70.81%和25.10%,纤维素损失率仅7.56%。与硫酸、盐酸预处理相比,氨基磺酸的半纤维素和木质素去除率不如硫酸、盐酸预处理,但固体回收率更高,纤维素损失率低,能保留更多纤维素有效成分。进一步酶解显示,氨基磺酸预处理的纤维素转化率高于硫酸、盐酸预处理。氨基磺酸作为一种新的酸预处理试剂,在木质纤维素降解上有良好应用前景。     

Internal structure of the starch granule revealed by AFM

Atomic force microscopy images of sectioned native corn starch granules show evidence of the well-known radial organisation of the starch macromolecules, with the less-ordered hilum region near to the centre. Native granules show blocks 400-500 nm in size that span the growth rings. Lintnerised starch granules, where a mild acid hydrolysis has been used to remove the amorphous and less crystalline parts of the granule, clearly show smaller 'blocklets' within the rings approximately 10-30 nm in size. This level of organisation within the growth rings corresponds to the blocklet or superhelix structures that have been proposed in the literature for the association or clustering of amylopectin helices. Mechanical property imaging techniques have provided enhanced contrast to view this morphology, and shown the deformability of the starch structure under contact mode imaging conditions.     

Reconstruction of lignin and hemicelluloses by aqueous ethanol anti‐solvents to improve the ionic liquid‐acid pretreatment performance of Arundo donax Linn

Ionic liquid (IL)‐acid pretreatment is known to not only enhance the enzymatic hydrolysis efficiency of lignocellulose but also to generate deposits on the surface of fiber by conventional water regeneration, which retard the increment. In this study, ethanol aqueous solution regeneration was developed as a new method to change the substrates characteristics for IL‐acid pretreatment and their effects on the enzymatic hydrolysis were evaluated. Following the IL‐acid reaction, the biomass slurry was subjected to ethanol aqueous solution at various concentration. Results indicated that anti‐solvent choice significantly influenced the reconstruction of both hemicelluloses and lignin as a result of the competition between water and ethanol. The partial removal of hemicelluloses and suitable lignin re‐localization contributed to a more porous structure. Consequently, the cellulose digestibility of aqueous ethanol regenerated samples was dramatically enhanced to ～100% and approximately 11‐ and 2‐fold higher than that of untreated and conventional water regenerated pretreated samples, respectively. A giant leap in the initial rate of enzymatic hydrolysis was also detected in 50% ethanol aqueous solution regenerated samples and only about 10 hr was needed to convert 80% of cellulose to glucose due to the appearance of cellulose II hydrate‐like and more porous structure.     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.