Nanoscale Structure of the Cell Wall Protecting Cellulose from Enzyme Attack (original) (raw)
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Industrial Crops and Products, 2019
Pretreatment is an important technological step for the lignocellulose conversion into fuels and biochemicals. Steam explosion (STEX) pretreatment is a fast method for affecting of plant cell wall architecture by a sharp pressure change at high temperatures. Here, eight different STEX conditions were applied to sugarcane bagasse (SCB) samples using auto-catalyzed and sulfuric acid catalyzed processes under a range of combined severity factors (CSFs; from 0.37 to 1.34). Our results showed that STEX of auto-catalyzed and sulfuric acid-catalyzed SCB led to the removal of 70-80 % and 82-92 % of xylose, respectively. Enzymatic hydrolysis of auto-catalyzed and sulfuric acid catalyzed bagasse was performed of washed and un-washed pretreated material to access the impact of soluble inhibitors on overall cellulosic sugars recovery. Auto-catalyzed bagasse after washing showed significantly higher glucan conversion (96%) than washed bagasse after pretreatment (62%). On the other hand, sulfuric acid-catalyzed bagasse after washing had 94% glucan conversion as opposed to unwashed material which again had significantly smaller conversion levels (73%). Physical properties and chemical composition were characterized using high performance liquid chromatography, X-ray diffraction, 13 C solid-state nuclear magnetic resonance and confocal laser scanning microscopy. The obtained results contribute to understanding of an influence of the morphological changes resulting from pretreatments on the efficiency of enzymatic hydrolysis. We show that the changes in physical structure and chemical composition of the pretreated biomass might not be determined solely by combined severity factor of the pretreatments, but by the whole complexity of chemical modifications that different conditions of pretreatments introduce in the plant biomass samples.
Acid Hydrolysis of Cellulose. Part I. Experimental Kinetic Analysis
Acid Hydrolysis of Cellulose. Part I. Experimental Kinetic Analysis, 1993
The main objective of this investigation is to obtain experimental data for the sulfuric acid hydrolysis of cotton and mechanically pretreated cotton fibres. These data indicate that some glycosidic bonds of cellulose have very high accessibility to catalytic ions. It was also shown that milling increases the accessibility of some glycosidic bonds of cellulose and decreases the volume of the crystalline regions of cotton. From the glucose yield versus time data, it was found that the effect of milling on the rate of cellulose depolymerization depends on the reactivity and accessibility of the glycon rings of cellulose. It was also found that at 1OO " C, the rate of cellulose depolymerization was not affected by the extraction of cotton wax and this was related to a rolling up process of cotton wax caused by melting. The kinetic constants of glucose degradation and cellobiose hydrolysis have been determined for the stochastic simulation of cellulose depolymerization which is the subject of the second part of this work. L'objectif principal de ce travail est l'obtention de donnCes exp6rimentales sur l'hydrolyse acide (sulfurique) des fibres du coton nature1 et du coton pretraitt mtcaniquement. Les rCsultats obtenus montrent que certaines liaisons glycosi-diques de la cellulose prksentent une grande accessibilitk aux ions catalytiques. Nous avons aussi montrC qu'un pretraitement mCcanique augmente I'accessibilite de certains liens glycosidiques de la cellulose et diminue le volume des regions cristallines du coton. Les donnCes exPCrimentales du glucose produit en fonction du temps, indiquent que les effets d'un pritraitement mtcanique sur la vitesse de dipolymerisation de la cellulose dCpendent de la rkactivitd et de l'acces-sibilitC des glycones de la cellulose. Nous avons aussi constat6 qu'il 1OO " C, la vitesse de d6polymirisation de la cellulose n'est pas affectee par l'extraction des cires du coton. Nous avons relit5 celil il une globulisation des cires causCe par leur fusion. Les constantes cinktiques de la degradation du glucose et de l'hydrolyse du cellobiose ont CtC deter-minees pour la simulation stochastique de la ddpolymerisation de la cellulose qui fera l'objet de la seconde partie de ce travail.
Cellulose accessibility determines the rate of enzymatic hydrolysis of steam-pretreated spruce
Bioresource Technology, 2012
h i g h l i g h t s " Degradability and surface characteristics of steam-pretreated spruce were studied. " A clear correlation between rate of enzymatic hydrolysis and BET area was found. " Higher pretreatment severity resulted in a more accessible cellulose fraction. " The influence by cellulose accessibility was larger than by surface lignin content.
The effect of enzyme concentration on the rate of the hydrolysis of cellulose
Biotechnology and Bioengineering, 1989
The relationship among extent of hydrolysis, reaction time, and enzyme dosage was investigated. For this, Sigmacell 50 and pretreated poplar wood (20 g/L) was hydrolyzed with varying dosages of cellulases from three different sources (5 to 100 FPU/g) for time periods ranging from 2 to 94 h. It was found that the formation of glucose can be described by summation of two parallel first order reactions. The extent of hydrolysis at fixed time increases with increasing enzyme dosage in a hyperbolic function. From the empirical data it is possible to calculate the fractions of easily and difficult hydrolyzable cellulose and the digestability which could maximally be obtained at infinite enzyme loadings. In the system Sigmacell 50 and Celluclast the easily and difficult hydrolyzable components are 43.0 and 57.0%, respectively, and the maximum digestability at 94 h i s 82.6%. Poplar wood, steam treated at 200°, 220", and 240°C, showed with Celluclast at 24 h a maximum digestability (weight percentage of wood degraded to glucose) of 43.9, 64.9, and 68.0%. The relationships derived from experimental data allow one to compare objectively the effectiveness of different cellulase enzymes and different pretreatments.
Biotechnology for Biofuels
Background: Steam explosion pretreatment has been examined in many studies for enhancing the enzymatic digestibility of lignocellulosic biomass and is currently the most common pretreatment method in commercial biorefineries. The information available about the effect of the explosive decompression on the biochemical conversion is, however, very limited, and no studies prove that the latter is actually enhanced by the explosion. Hence, it is of great value to discern between the effect of the explosion on the one hand and the steaming on the other hand, to identify their particular influences on enzymatic digestibility. Results: The effect of the explosive decompression in the steam explosion pretreatment of spruce wood chips on their enzymatic cellulose digestibility was studied systematically. The explosion had a high influence on digestibility, improving it by up to 90 % compared to a steam pretreatment without explosion. Two factors were identified to be essentially responsible for the effect of the explosion on enzymatic digestibility: pretreatment severity and pressure difference of the explosion. A higher pretreatment severity can soften up and weaken the lignocellulose structure more, so that the explosion can better break up the biomass and decrease its particle size, which enhances its digestibility. In particular, increasing the pressure difference of the explosion leads to more defibration, a smaller particle size and a better digestibility. Though differences were found in the micro-and nanostructure of exploded and nonexploded biomass, the only influence of the explosion on digestibility was found to be the macroscopic particle size reduction. Steam explosion treatments with a high severity and a high pressure difference of the explosion lead to a comparatively high cellulose digestibility of the-typically very recalcitrant-softwood biomass. Conclusions: This is the first study to show that explosion can enhance the enzymatic digestibility of lignocellulosic biomass. If the enhancing effect of the explosion is thoroughly exploited, even very recalcitrant biomass like softwood can be made enzymatically digestible.
Biotechnology Progress, 2008
Steam-exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam-explosion pretreatment process. The SE temperature and time conditions were 214°C and 6 min, resulting in a log R 0 of 4.13. In enzymatic hydrolysis tests at 45°C, the biomass loading in the bioreactor was 100 g DW /L (dry weight) and the enzyme-to-biomass ratio 0.06 g/g DW. The enzyme activities for endo-glucanase, exo-glucanase, and-glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5-hydroxymethyl furfural (5-HMF), syringaldehyde, 4-hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5-HMF, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5-HMF (0.5 g/L) in SSF of 100 g DW /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.
Industrial Crops and Products, 2020
Significant improvements in pretreatments and a deeper understanding of changes in the physical structure and chemical composition of pretreated lignocellulosic materials are required to guarantee the technical and economic sustainability of second-generation ethanol technologies. In present work, several biophysical techniques were applied to characterize sugarcane bagasse samples that have been subjected to autocatalytic, phosphoric acid and sulfuric acid catalyzed steam explosion under equivalent combined severity factors (CSF). Confocal laser scanning microscopy and fluorescence lifetime imaging studies demonstrated unequal changes in lignin distribution in the plant cell wall that could be nicely correlated with substrate susceptibility to enzymatic hydrolysis. Furthermore, solid state nuclear magnetic resonance corroborated these observations by showing the differences in biomass composition primarily associated with hemicellulose removal, whereas X-ray diffraction analysis revealed changes in crystallinity indexes and average crystallite sizes. Thus, the impact of pretreatment temperature on the physical structure and chemical composition of CSF-equivalent steam-exploded sugarcane bagasse was identified as the key factor for developing substrate accessibility. This calls for caution in using combined severity factor as a chief parameter for comparison of different pretreatment conditions.
Biomass Feedstock Pre-Processing – Part 1: Pre-Treatment
Biofuel's Engineering Process Technology, 2011
2. Lignocellulosic biomass characterization 2.1 Structure of lignocellulosic material Lignocellulosic material refers to plant biomass that is composed of cellulose, hemicellulose, and lignin (Fig. 2) (Lin and Tanaka, 2006). The major combustible component of non-food energy crops is cellulose, followed by lignin. Cellulose: Cellulose is an organic polysaccharide consisting of a linear chain of several hundreds to over nine thousand β(1→4) linked D-glucose (C 6 H 10 O 5)n units (Crawford, 1981; Updegraff, 1969). Cellulose, a fibrous, tough, water-insoluble substance, is found in the cell walls of plants, particularly in the stalks, stems, trunks and all the woody portions of the plant body (Nelson and Cox, 2005). Cellulose comprises 40-60% of the dry weight of plant material (the cellulose content of cotton is 90% and that of wood is 50%) (Encyclopaedia Britannica, 2008; USDE, 2006). Zandersons et al. (2004) and Shaw (2008) reported that binding of wood material during hot pressing / densification is mainly dependent on the transition of cellulose into the amorphous state. According to Hon (1989), due to the semi-crystalline structure, hydrogen bonded cellulose cannot be dissolved easily in conventional solvents, and it cannot be melted before it burns; hence, cellulose itself is not a suitable adhesive. This can be overcome by breaking the hydrogen bonds, thus making the cellulose molecule more flexible (Hon 1989). Cellulose requires a temperature of 320°C and pressure of 25 MPa to become amorphous in water (Deguchi et al., 2006). Hemicellulose: Hemicellulose is made of several heteropolymers (matrix polysaccharides) present in almost all plant cell walls along with cellulose (Fig. 2). While cellulose is crystalline, strong, and resistant to hydrolysis; hemicellulose has a random, amorphous structure with less strength. Hemicellulose is a polysaccharide related to cellulose and comprises 20-40% of the biomass of most plants. In contrast to cellulose, hemicellulose is derived from several sugars in addition to glucose, including especially xylose but also mannose, galactose, rhamnose and arabinose (Shambe and Kennedy, 1985). Branching in hemicellulose produces an amorphous structure that is more easily hydrolyzed than cellulose (Shaw, 2008). Also, hemicellulose can be dissolved in strong alkali solutions. Hemicellulose provides structural integrity to the cell. Some researchers believe that natural bonding may occur due to the adhesive properties of degraded hemicellulose (Bhattacharya et al., 1989). Lignin: Lignin is a complex chemical compound most commonly derived from wood and is an integral part of the cell walls of plants (Lebo et al., 2001; Zandersons et al., 2004). The compound has several unusual properties as a biopolymer, not the least its heterogeneity in lacking a defined primary structure. Lignin fills the spaces in the cell wall between cellulose and hemicellulose (Fig. 2). It is covalently linked to hemicellulose and thereby crosslinks different plant polysaccharides, conferring mechanical strength to the cell wall and consequently to the whole plant structure (Chabannes et al., 2001). Lignin acts as a binder for the cellulose fibres (Fig. 2). van Dam et al. (2004) have reported that lignin can be used as an intrinsic resin in binderless board production due to the fact that when lignin melts (temperatures above 140°C), it exhibits thermosetting properties. Lignin is the component that permits adhesion in the wood structure, and is a rigidifying and bulking agent (Anglès et al., 2001). Lehtikangas (2001) reported that water (8-15%) in pellets will reduce the softening temperature of lignin to 100-135°C by plasticizing the molecular chains. The adhesive properties of thermally softened lignin are thought to contribute considerably to the strength characteristics of briquettes made of lignocellulosic materials (Granada et al., 2002; Shaw, 2008).
The scarcity and high price associated with fossil fuel has urged countries to research resources for alternative energy sources. Biofuels like bioethanol produced from lignocellulosic biomass (corn cob) were considered potential alternative. Cellulose composition from isolated cell wall material of corn cobs was investigated under two different pre-treatments using H2SO4 and NH4OH at varying concentrations of 5%, 10%, 20%, 30% and 40%. Cell wall not treated acted as control. Colorimetric anthrone-assay followed by absorbance reading at 625nm revealed that glucose is present in reasonable amount in corn cob. The analysis of variance (ANOVA) indicated significant differences among pre-treated compared to untreated (Control) corn cob samples at p≤0.05. Acid pretreatment showed better glucose yield compared to alkali pre-treatment with results revealing 20% (19.37µg/ml) H2SO4 to be the optimal concentration producing highest glucose yield. The study reveals the potential of corn cob as a lignocellulosic feed stock for biofuel production. Historic