Hydrolysis of steam-pretreated lignocellulose

The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40 degrees C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4 degrees C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40 degrees C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40 degrees C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.     

Quantitative assessment of the enzymatic degradation of amorphous cellulose by using a quartz crystal microbalance with dissipation monitoring

The systematic evaluation of the degradation of an amorphous cellulose film by a monocomponent endoglucanase (EG I) by using a quartz crystal microbalance with dissipation monitoring (QCM-D) identified several important aspects relevant to the study the kinetics of cellulose degradation by enzymes. It was demonstrated that, to properly evaluate the mechanism of action, steady state conditions in the experimental set up need to be reached. Rinsing or diluting the enzyme, as well as concentration of the enzyme, can have a pronounced effect on the hydrolysis. Quantification of the actual hydrolysis was carried out by measuring the film thickness reduction by atomic force microscopy after the enzymatic treatment. The values correlated well with the frequency data obtained by QCM-D measurement for corresponding films. This demonstrated that the evaluation of hydrolysis by QCM-D can be done quantitatively. Tuning of the initial thickness of films enabled variation of the volume of substrate available for hydrolysis which was then utilized in establishing a correlation between substrate volume and hydrolytic activity of EG I as measured by QCM-D. It was shown that, although the amount of substrate affects the absolute rate of hydrolysis, the relative rate of hydrolysis does not depend on the initial amount of substrate in steady state system. With this experimental setup it was also possible to demonstrate the impact of concentration on crowding of enzyme and subsequent hydrolysis efficiency. This effort also shows the action of EG I on a fully amorphous substrate as observed by QCM-D. The enzyme was shown to work uniformly within the whole volume of swollen film, however being unable to fully degrade the amorphous film.     

Exopolysaccharide production by a marine cyanobacterium Cyanothece sp.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.     

Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.     

Structural changes in wood during ozonation

It is found that ozone treatment of aspen wood leads to changes in its structural characteristics, i.e., its specific surface area and the crystallinity index of cellulose. Using optical microscopy, it is shown that ozonation is accompanied by a decrease in the average size and visible surface of wood particles. The values for the specific area of the outer surface of samples are calculated. The specific surface area available to the enzyme molecules is determined from data on the adsorption of inert protein hemoglobin on wood. It is shown that this value is an order of magnitude higher than that of the outer surface and increases considerably for an ozonized sample. Based on the results from X-ray analysis, it is established that the structure of cellulose is disordered during ozone delignification, as is indicated by a reduction in the crystallinity index and crystallite sizes.     

Smooth model cellulose I surfaces from nanocrystal suspensions

Removal of lignin, hemicelluloses and other minor components during pulping results in a porous fibrillar structure. Interactions of cellulose fibre surfaces with wet-end additives and other materials depend both on the interfacial properties of the cellulose and on the morphology of the surface. It would be useful to be able to separate the interactions with the cellulose from those that depend on surface roughness and porosity by preparing flat cellulose surfaces. Current methods give surfaces of amorphous cellulose or of cellulose II, differing in density and crystallinity from the original cellulose I surface. We propose a new route to prepare smooth model surfaces of cellulose I, starting from colloidal dispersions of cellulose I nanocrystals. The nanometer-sized width of these rod-like colloidal particles allows a relatively flat surface to be prepared from the suspension by casting an aqueous suspension on an appropriate surface and allowing the water to evaporate. Oriented films can be prepared by spin-coating or shearing. The surface composition and morphology of the films were examined by X-ray photoelectron spectroscopy and atomic force microscopy.     

Flame-retarded hydrophobic cellulose through impregnation with aqueous solutions and supercritical CO2

We have developed flame-retarded hydrophobic cellulose-based materials by producing in situ water-soluble and insoluble inorganic microparticles on various surfaces of native cellulose (filter paper and pure cotton textile). The nanoparticles were produced by simple impregnation of cellulose with two different aqueous solutions followed by a third impregnation with supercritical CO₂. Finally, the composite cellulose materials were covered by a silicon-based polymer thin film, to turn it into hydrophobic and prevent the water-soluble particles from absorbing humidity. The obtained flame-retardant behaviour is due to a combination of mechanisms. The total treatment of cellulose has an impact on, both its surface morphology and its hydrophilicity. Thus, the hydrophobic nature of the silicon-based polymer film along with the roughness caused by the presence of the inorganic particles and the inherent roughness of native cellulose resulted in superhydrophobic behaviour. The same process-concept was also applied to regenerated (from newspaper) cellulose with ionic liquids. The produced materials were characterised by thermogravimetric analysis, differential scanning calorimetry, infrared spectroscopy, scanning electron microscopy and water contact angle measurements.     

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase from Trichoderma viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.     

Cellulose loading and water sorption value as important parameters for the enzymatic hydrolysis of cellulose

The enzymatic hydrolysis of cotton raw cellulose (RC) samples, sieved RC samples through meshes <100 (CS1), 100–200 (C12), 200–400 (C24), mercerized RC samples (M-C), freeze-dried RC (RC-FD) samples, microcrystalline cellulose Avicel, bacterial cellulose (BC), raw sisal pulp and mercerized sisal pulp (S-M) was performed at cellulose-to-cellulase mass ratios of 1,000:1, 699:1, 400:1, 100:1 and 10:1. The index of crystallinity and water sorption values were quantified for all samples. The morphological features were analyzed by means of scanning electron microscopy (SEM). For cellulose-to-cellulase mass ratio of 100:1 and 10:1, the maximum hydrolysis extents of cellulose samples after 24 h reaction could not be correlated with their physical characteristics. However, hydrolyses of samples with large water sorption values were faster than those with lower water sorption values. The hydrolysis efficiency decreased when the cellulose-to-cellulase mass ratio was greater than 400:1; under this condition a remarkable dependence of the hydrolysis yield on the type of cellulosic sample was observed. The water sorption ability could be directly correlated with the hydrolysis extent, except for RC-FD and BC samples, which presented the lowest values. In the former, freeze-drying has led to pore collapse, with concomitant reduction of the amount of adsorbed water. For the latter sample, the densely packed structure made the water sorption slower than in all other samples. Despite of this fact, the presence of nanofibrils on the surface of BC (as detected by SEM) improved the enzyme adsorption, indicating that analysis by complementary techniques should be performed in order to predict the enzymatic hydrolysis efficiency.     

Evaluation of liquid ammonia treatment on surface characteristics of hemp fiber

In this study, we investigated the effects of liquid ammonia treatment on the surface characteristics of hemp fibers. We determined the elemental composition, morphological structure, roughness, and wettability of fiber surface using techniques such as electron spectroscopy for chemical analysis, scanning electron microscopy, atomic force microscopy, and contact angle measurements. The lignin coverage on the hemp surface was calculated from the O/C ratio and the C₁ content. The results show that lignin removal from the fiber surface was significantly greater than that from the fiber bulk. After the treatment, the O/C ratio of hemp fibers increased, and cellulose was exposed. The proportion of O₂ species that contributed to formation of hydrogen bonds increased; this further increased the number of hydrophilic groups in the hemp fibers, improving the fiber wettability. The liquid ammonia treatment did not change the large dislocation structures in hemp fibers, but the removal of noncellulosic materials from the fiber surface increased the roughness of the fiber surface.     

Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the "overall hydrolysis rates" with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the "primary hydrolysis step" (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster "secondary hydrolysis" (conversion of soluble oligomers to glucose).     

Using of silica particles as porogen for preparation of macroporous chitosan macrospheres suitable for enzyme immobilization

Porous chitosan macrospheres were prepared at the first time by using silica particles as porogen, and the optimal weight ratio of silica to chitosan during preparation was determined. Scanning electron microscopy micrographs showed that the support with silica as porogen (support I) had a much more porous surface structure than the support without porogen (support II). Both supports were applied to immobilize β-galactosidase from Aspergillus oryzae. Much higher specific activity and yield of galactose hydrolysis products were observed for the support I. Properties of both immobilized enzyme were determined and compared with the free enzyme, satisfactory results were obtained in thermostability, pH arid operational stability, Michaelis constants K _m and in maximum velocity of hydrolysis (V _m). Suggested method allow to prepare chitosan macrospheres as immobilized enzyme carrier with moreporous surface structure and more active reaction groups.     

Savinase proteolysis of insulin Langmuir monolayers studied by surface pressure and surface potential measurements accompanied by atomic force microscopy (AFM) imaging

The mechanism of the enzymatic action of Savinase on an insulin substrate organized in a monolayer at the air-water interface was studied. We followed two steps experimental approach classical surface pressure and surface potential measurements in combination with atomic force microscopy imaging. Utilizing the barostat surface balance, the hydrolysis kinetic was followed by measuring simultaneously the decrease in the surface area and the change of the surface potential versus time. The decrease in the surface area is a result of the random scission of the peptide bonds of polypeptide chain, progressively appearance of amino acid residues, and their solubilization in the aqueous subphase. The interpretation of the surface potential data was based on the contribution of the dipole moments of the intact and broken peptide groups which remain at the interface during the proteolysis. An appropriate kinetic model for the Savinase action was applied, and the global kinetic constant was obtained. The application of the AFM revealed the state of the insulin monolayers before and after the Savinase action. The comparison of the topography of the films and the roughness analysis showed that insulin Langmuir-Blodgett (LB) films transferred before the enzyme action were flat, while at the end of hydrolysis, roughness of films has increased and the appearance of 3D structures was observed.     

In-situ growth of silica nanoparticles on cellulose and application of hierarchical structure in biomimetic hydrophobicity

Monodispersed silica nanoparticles were prepared by a simple two-step method with hydrolysis and condensation. The materials were characterized by dynamic light scattering (DLS), SEM and TEM. Through in-situ growth of silica nanoparticles on cotton fabrics, a dual-scaled surface with nanoscaled roughness of silica and microscaled roughness of cellulose fiber was generated. After the modification of the low surface energy, the wettability of smooth silicon slide, silicon slide with nanoscaled roughness of silica particles, cotton fabric, and cotton fabric with silica particles was evaluated by the tests of the contact angle (CA) and the advancing and receding contact angle (ARCA). The cotton fabric with dual-scaled roughness exhibits a static CA of 149.8° for 4 μL water droplet and a hysteresis contact angle (HCA) of 1.8°. The results of CA and HCA show that microscaled roughness plays a more important role than nanoscaled roughness for the value of CA and HCA. The results in the hydrostatic pressure test and the rain test show the important contribution of nanoscaled roughness for hydrophobicity.     

Rotation of Cellulose Ribbons During Degradation with Fungal Cellulase

Degradation of bacterial cellulose with a commercial cellulase, Celluclast 1.5L (Novo Nordisk), from the fungus Trichoderma reesei, causes a rotational movement of the cellulose microfibrils. Purified cellulases (CBH I, CBH II, and EG II) do not induce rotation of bacterial cellulose, however, ratios of CBH I and EG II do cause rotation of bacterial cellulose. Equimolar amounts of CBH I or CBH II and EG II do not result in motion during degradation. Based on these observations, we provide further evidence supporting, at least on theoretical grounds, the hypothesis that cellulose chains have intrinsic chirality. As the cellulase enzymes interact with and degrade the cellulose fibrils, the crystalline structure of the cellulose is altered, allowing the linear cellulose polymers to relax into a lower energy state, thus relieving the strain induced by crystallization of the nascent -glucan chains during the biogenesis of the microfibril. This conversion of crystalline bacterial ribbons into more relaxed conformations produces the rotation observed during the treatment of bacterial cellulose with cellulase.     

The effect of Trichoderma reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp

Four purified cellulases, a xylanase and mannanase from Trichoderma reesei were used to treat never-dried bleached pine kraft pulp prior to refining, and the effects on pulp properties were evaluated. The enzymatic treatments hydrolysed up to 0.8% of pulp dry weight. The results demonstrated that the individual cellulases have profoundly different modes of action in modifying pulp carbohydrates. This is especially clear when comparing their effects at the same level of hydrolysis. Pretreatment with cellobiohydrolases I (CBH I) and II (CBH II) had virtually no effect on the development of pulp properties during refining, except for a slight decrease in strength properties. On the contrary, endoglucanase I (EG I) and endoglucanase II (EG II) improved the beatability of the pulp as measured by Schopper--Riegler value, sheet density and Gurley air resistance. Of the endoglucanases, EG II was most effective in improving the beating response. The combinations of CBH I with EG I and EG II had similar effects on the pulp properties as the endoglucanases alone, although the amount of hydrolysed cellulose was increased. Pretreatments with xylanase or mannanase did not appear to modify the pulp properties. The same enzyme treatments which improved the beatability, however, slightly impaired the pulp strength, especially tear index at the enzyme dosages used. When compared at a given level of cellulose hydrolysis, the negative effect of EG II on strength properties was more pronounced compared with EG I. Thus, the exploitation of cellulases for fibre treatments requires careful optimization of both enzyme composition and dosage. Since the endoglucanases had no positive effect on the development of tensile strength, it is suggested that the explanation for the increased beating response is increased fibre breakage and formation of fines, rather than improved flexibilization. This revised version was published online in August 2006 with corrections to the Cover Date.     

Superhydrophobic cellulose nanocomposites

Superhydrophobic cellulose nanocomposites were prepared using a multi-step nanoengineering process. The combination of different techniques made it possible to construct novel features at the ensuing surface, characterized by both an increase in its roughness induced by amorphous silica particles and a reduction in its energy insured by perfluoro moieties, giving rise to water contact angles approaching 150 degrees . The modification calls upon an aqueous LbL system followed by siloxane hydrolysis, both conducted at room temperature in air. Each modification was followed by scanning electron microscopy (SEM) and atomic force microscope (AFM). These original cellulose-silica-silane composite materials open the way to further valorisations of a ubiquitous renewable resource in applications such as water repellence and self-cleaning.     

Probing the active sites for CO dissociation on ruthenium nanoparticles

The active sites for CO dissociation were probed on mass-selected Ru nanoparticles on a HOPG support by temperature programmed desorption spectroscopy using isotopically labelled CO. Combined with transmission electron microscopy we gain insight on how the size and morphology of the nanoparticles affect the CO dissociation activity. The Ru nanoparticles were synthesized in a UHV chamber by gas-aggregation magnetron sputtering in the size range from 3 to 15 nm and the morphology was investigated in situ by scanning tunneling microscopy and ex situ by high resolution transmission electron microscopy. Surprisingly, it was found that larger particles were more active per surface area for CO dissociation. It is suggested that this is due to larger particles exposing a more rough surface than the smaller particles, giving rise to a higher relative amount of under-coordinated adsorption sites on the larger particles. The induced surface roughness is proposed to be a consequence of the growth processes in the gas-aggregation chamber.     

Physical properties of TEMPO-oxidized bacterial cellulose nanofibers on the skin surface

Water-dispersed bacterial cellulose nanofibers were prepared via an oxidation reaction using 2,2,6,6-tetramethyl-1-piperidine-N-oxy radical (TEMPO) as a catalyst. It was found that TEMPO-oxidized bacterial cellulose nanofibers (TOCNs) synthesized via sodium bromide-free methods are similar to those synthesized using sodium bromide. The TOCNs retained their unique structure in water as well as in emulsion. TOCNs adhere to the skin surface while maintaining nanofibrous structures, providing inherent functions of bacterial cellulose, such as high tensile strength, high water-holding capacity, and blockage of harmful substances. When gelatin gels as model skin were coated with TOCNs, the hardness representing the elasticity was increased by 20% compared to untreated gelatin gel because TOCNs could tightly hold the gelatin structure. When porcine skin was treated with TOCNs, carboxymethyl cellulose, and hydroxyethyl cellulose, the initial water contact angles were 26.5°, 76.5°, and 64.1°, respectively. The contact angle of TOCNs dramatically decreased over time as water penetrated the fibrous structure of the TOCN film. When observed by scanning electron microscopy and confocal microscopy, TOCNs on the skin surface provided physical gaps between particles and the skin, blocking the adsorption of particulate matter to the skin surface. On the contrary, the structure of water-soluble polymers was disrupted by an external environment, such as water, so that particulate matter directly attached to the skin surface. Characterization of TOCNs on the skin surface offered insight into the function of nanofibers on the skin, which is important for their applications with respect to the skin and biomedical research.     

Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose

This paper investigates the enzymatic hydrolysis of three main allomorphic forms of microcrystalline cellulose using different cellulases, from Trichoderma reesei and from Aspergillus niger, respectively. It was demonstrated that both the morphological and crystalline structures are important parameters that have a great influence on the course of the hydrolysis process. The efficiency of the enzymatic hydrolysis of cellulosic substrates was estimated by the amounts of reducing sugar and by the yield of the reaction. Changes in the average particle sizes of the cellulose allomorphs were determined during enzymatic hydrolysis. The accumulation of soluble sugar within the supernatant was used as a measure of the biodegradation process’s efficiency, and was established by HPLC-SEC analysis. Any modifications in the supramolecular structure of the cellulosic residues resulting from the enzymatic hydrolysis were determined by X-ray diffraction. The action of each cellulase was demonstrated by a reduction in the crystalline index and the crystallite dimensions of the corresponding allomorphic forms. The crystalline structure of allomorphic forms I and II did not suffer significant modifications, while cellulose III recorded a partial return to the crystalline structure of cellulose I. The microstructures of cellulose allomorph residues were presented using optical microscopy and scanning electron microscopy.