Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce

Background

The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs).

Results

LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis.

Conclusions

LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

     

Production of sugars from wood using high-pressure hydrogen chloride

Saturating wood particles with HCl gas under pressure was found to be an effective pretreatment prior to subjecting wood to dilute acid hydrolysis. Pretreament is necessary to release sugars from wood because of the tight lattice structure of cellulose. The HCl gas makes the cellulose more susceptible to subsequent acid hydrolysis and the glucose yield is doubled when dilute acid hydrolysis is preceded by HCl saturation at high pressure. The saturation was most effectively performed in a fluidized bed reactor, with pure HCl gas fluidizing an equal volume of ground wood plus inert particles. The fluidized bed effectively dissipated the large amount of heat released upon HCl absorption into the wood. Batch reaction times of 1 h at 315 psia gave glucose yields of 80 degrees and xylose yields of 95 degrees after dilute acid hydrolysis. A model was developed which proposed gas diffusing through the solid as limiting the reaction rate and this was found to effectively describe the HCl-wood reaction in the fluidized bed. The HCl was found to form a stable adduct with the lignin residue in the wood, in a ratio of 3.33 mole of lignin monomer. The adduct was broken upon the addition of water.     

Does elevated atmospheric CO2 concentrations affect wood decomposition?

This study was conducted to test the hypothesis that wood tissues generated under elevated atmospheric [CO₂] have lower quality and subsequent reduced decomposition rates. Chemical composition and subsequent field decomposition rates were studied for beech (Fagus sylvatica L.) twigs grown under ambient and elevated [CO₂] in open top chambers. Elevated [CO₂] significantly affected the chemical composition of beech twigs, which had 38% lower N and 12% lower lignin concentrations than twigs grown under ambient [CO₂]. The strong decrease in N concentration resulted in a significant increase in the C/N and lignin/N ratios of the beech wood grown at elevated [CO₂]. However, the elevated [CO₂] treatment did not reduce the decomposition rates of twigs, neither were the dynamics of N and lignin in the decomposing beech wood affected by the [CO₂] treatment, despite initial changes in N and lignin concentrations between the ambient and elevated [CO₂] beech wood. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conversion of Japanese red pine wood (Pinus densiflora) into valuable chemicals under subcritical water conditions

A comparative study on the decomposition of Japanese red pine wood under subcritical water conditions in the presence and absence of phosphate buffer was investigated in a batch-type reaction vessel. Since cellulose makes up more than 40-45% of the components found in most wood species, a series of experiments were also carried out using pure cellulose as a model for woody biomass. Several parameters such as temperature and residence time, as well as pH effects, were investigated in detail. The best temperature for decomposition and hydrolysis of pure cellulose was found around 270 °C. The effects of the initial pH of the solution which ranged from 1.5 to 6.5 were studied. It was found that the pH has a considerable effect on the hydrolysis and decomposition of the cellulose. Several products in the aqueous phase were identified and quantified. The conditions obtained from the subcritical water treatment of pure cellulose were applied for the Japanese red pine wood chips. As a result, even in the absence of acid catalyst, a large amount of wood sample was hydrolyzed in water; however, by using phosphate buffer at pH 2, there was an increase in the hydrolysis and dissolution of the wood chips. In addition to the water-soluble phase, acetone-soluble and water-acetone-insoluble phases were also isolated after subcritical water treatment (which can be attributed mainly to the degraded lignin, tar, and unreacted wood chips, respectively). The initial wood:acid ratio in the case of reactions catalyzed by phosphate buffer was also investigated. The results showed that this weight ratio can be as high as 3:1 without changing the catalytic activity. The size of the wood chips as one of the most important experimental parameters was also investigated.     

Degradation of polysaccharides and lignin in wood ozonation

Ozonation has been considered as a method for the pretreatment of plant biomass to obtain cellulose and monosaccharides. Ozone consumption by aspen wood with various moisture contents has been investigated. We have considered the gradual transformation of the substrate: wood to ozonated wood to cellulose-containing product (CP) to holocelluloze (HC) and to cellulose. Yields of ozonated wood (OW), the (CP), water-soluble ozonation products, HC, and cellulose have been determined. The lignin content in the CP has been estimated. Both HC and cellulose samples have been studied by IR spectroscopy. The degree of polymerization and molecular mass distribution of cellulose obtained from ozonated wood have been determined. It has been shown that wood destruction by ozone is accompanied by degradation of lignin, hemicelluloses, and cellulose.It has been found that physicochemical properties of cellulose obtained from ozonated wood can be regulated by the variation of the initial moisture content in the substrate. Both molecular ozone and radical species, which are generated in the course of ozone reactions with water present in the substrate structure, participate in wood destruction.     

A sustainable woody biomass biorefinery

Woody biomass is renewable only if sustainable production is imposed. An optimum and sustainable biomass stand production rate is found to be one with the incremental growth rate at harvest equal to the average overall growth rate. Utilization of woody biomass leads to a sustainable economy. Woody biomass is comprised of at least four components: extractives, hemicellulose, lignin and cellulose. While extractives and hemicellulose are least resistant to chemical and thermal degradation, cellulose is most resistant to chemical, thermal, and biological attack. The difference or heterogeneity in reactivity leads to the recalcitrance of woody biomass at conversion. A selection of processes is presented together as a biorefinery based on incremental sequential deconstruction, fractionation/conversion of woody biomass to achieve efficient separation of major components. A preference is given to a biorefinery absent of pretreatment and detoxification process that produce waste byproducts. While numerous biorefinery approaches are known, a focused review on the integrated studies of water-based biorefinery processes is presented. Hot-water extraction is the first process step to extract value from woody biomass while improving the quality of the remaining solid material. This first step removes extractives and hemicellulose fractions from woody biomass. While extractives and hemicellulose are largely removed in the extraction liquor, cellulose and lignin largely remain in the residual woody structure. Xylo-oligomers, aromatics and acetic acid in the hardwood extract are the major components having the greatest potential value for development. Higher temperature and longer residence time lead to higher mass removal. While high temperature (>200°C) can lead to nearly total dissolution, the amount of sugars present in the extraction liquor decreases rapidly with temperature. Dilute acid hydrolysis of concentrated wood extracts renders the wood extract with monomeric sugars. At higher acid concentration and higher temperature the hydrolysis produced more xylose monomers in a comparatively shorter period of reaction time. Xylose is the most abundant monomeric sugar in the hydrolysate. The other comparatively small amounts of monomeric sugars include arabinose, glucose, rhamnose, mannose and galactose. Acetic acid, formic acid, furfural, HMF and other byproducts are inevitably generated during the acid hydrolysis process. Short reaction time is preferred for the hydrolysis of hot-water wood extracts. Acid hydrolysis presents a perfect opportunity for the removal or separation of aromatic materials from the wood extract/hydrolysate. The hot-water wood extract hydrolysate, after solid-removal, can be purified by Nano-membrane filtration to yield a fermentable sugar stream. Fermentation products such as ethanol can be produced from the sugar stream without a detoxification step.     

The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar

The use of alcohol/water/catalyst mixtures to delignify wood allows the lignin to be recovered in a usable form while leaving the carbohydrate fraction relatively intact. The effects of temperature, reaction time, and the type of solvent and catalyst on the delignification of milled poplar wood were investigated. The lignin, cellulose, and hemicellulose composition of the pretreated material was measured for each treatment condition. In addition, the pretreated samples were subjected to enzymatic hydrolysis using the cellulases produced by the thermophilic bacterium Thermomonospora sp. YX. The extent of enzymatic hydrolysis was characterized using an empirical model, and the results were used to examine the effectiveness of the pretreatment.     

The role of xylanases and laccases on hexenuronic acid and lignin removal

The variation of the contents in hexenuronic acids (HexA) and lignin in Eucalyptus kraft pulp during sequences with the laccase–mediator treatment with or without xylanase pretreatment was studied. The laccase–HBT system (HBT: 1-hydroxybenzotriazole) initially oxidized lignin alone but altered cellulose in the pulp as well after some time. Once all accessible lignin was removed, the system acted on HexA. As a result, the laccase–mediator treatment reduced the HexA content of the pulp, especially if a xylanase pretreatment was applied before. A previously unseen effect was observed here: HexA removal was found to depend on the laccase and HBT doses, but not on the reaction time. In addition, the xylanase pretreatment was found to strongly boost the effects of the laccase–HBT system by facilitating their access to HexA without affecting the lignin content.     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.     

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.     

Ecology of coarse wood decomposition by the saprotrophic fungus <Emphasis Type="Italic">Fomes fomentarius</Emphasis>

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.     

Statistical correlation of spectroscopic analysis and enzymatic hydrolysis of poplar samples

Spectroscopic characterization of poplar wood samples with different crystallinity indices, lignin contents, and acetyl contents was performed to determine changes in the biomass spectra and the effects of these changes on the hydrolysis yield. The spectroscopic methods used were X-ray diffraction for determining cellulose crystallinity (CrI), diffuse reflectance infrared (DRIFT) for changes in C-C and C-O bonds, and fluorescence to determine lignin content. Raman spectroscopy was also used to determine its effectiveness in the determination of crystallinity and C-C and C-O bond changes in the biomass as a complement to better-known methods. Changes in spectral characteristics and crystallinity were statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that influence enzymatic conversion to monomeric sugars. In addition, the different spectroscopic methods were evaluated separately to determine the minimum amount of spectroscopic data needed to obtain accurate predictions. The principal component regression (PCR) model with only the DRIFT data gives the best correlation and prediction for both initial rate of hydrolysis and also the 72-h hydrolysis yield. The factor that most affects both the initial rate and the 72-h conversion is the O-H bond content of the sample, which directly relates to the breakage of structural carbohydrates into smaller molecules.     

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.     

Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression

The adsorption of cellulase on cellulose and a lignacious residue was examined by using cellulase from Trichoderma reesei, hardwood pretreated by dilute sulfuric acid under high pressure, and a lignacious residue prepared by a complete enzymatic hydrolysis of the pretreated wood. A significant amount of cellulase was found to adsorb on the lignacious residue during the hydrolysis of the pretreated wood. Hence, the adsorption of enzyme on the lignacious residue as well as cellulose must be taken into account in the development of the hydrolysis kinetics. It was found that the adsorption of enzyme on cellulose and on the lignacious residue could be represented by Langmuir type isotherms. The data show that the pretreatment at a higher temperature results in more enzyme adsorption on the cellulose fraction and less on the lignacious residue fraction. The relationship between the hydrolysis rate and the amount of enzyme adsorbed is discussed.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

The cellulose/lignin assembly assessed by molecular modeling. Part 1: adsorption of a threo guaiacyl beta-O-4 dimer onto a Ibeta cellulose whisker.

The assembly of the two major cell wall components, cellulose and lignin, were investigated at the atomistic scale using molecular dynamics simulations. To this end, a molecular model of a cellulose crystal corresponding to the allomorph Ibeta and exhibiting different surfaces was considered to mimic the carbohydrate matrix present in native wood cell wall. The lignin model compound considered here is a threo guaiacyl beta-O-4 dimer. The dynamical process of adsorption of the lignin dimer onto the different surfaces of the cellulose crystal was examined. The modes of association between the two constituents were analyzed; energies of adsorption of the dimer are calculated favorable and of the same order of magnitude on all sides of the cellulosic model, suggesting that the deposition of lignin precursors onto cellulose fibers is non-specific from an enthalpic point of view. Interestingly, geometrical characteristics and energetical details of the adsorption are surface-dependent. Computed data have underlined the predominant contribution of van der Waals interactions for adsorption onto the (200) face, as well as the major influence of H-bonding interactions in the dynamical process of adsorption onto (110) and (1-10) faces. A large number of adsorption sites have been identified and a noticeable "flat" geometry of adsorption of the lignin dimer has been observed, as a consequence of the stacking interactions between lignin aromatic rings and C-H groups of cellulose. Importantly, these dispersive interactions lead to a preferential parallel orientation of lignin aromatic rings relative to the cellulose surface, notably on the (200) face. Such a parallel orientation is consistent with previously reported experimental observations.