Supramolecular Structure - A Key Parameter for Cellulose Biodegradation

Summary: Three different cellulosic substrata, like microcrystalline cellulose, cotton cellulose and spruce dissolving pulp, were chosen for biodegradation. The kinetics of the enzymatic hydrolysis of these celluloses by Trichoderma reesei, has been investigated. The experiments proved the fact that both the morphological structure and the crystalline one are crucial to the process and the ratio of the reactions. In addition, in order to obtain the most accessible cellulose substratum it was studied the biodegradation of cellulose allomorphs of spruce dissolving pulp. The insoluble cellulose fraction remaining after enzymatic hydrolysis was examined by X-ray diffraction method and it was established the degree of crystallinity and the average crystallite size. The enzymatic degradation is also proved by the decrease in the degree of polymerization of hydrolyzed samples.     

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.     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Studies concerning the accessibility of different allomorphic forms of cellulose

The supramolecular architecture and the morphological structure of cellulose play an important role in its accessibility. In order to evaluate the effect of the crystalline form of organization on the accessibility, we selected cellulosic materials with significant variations in the aforementioned characteristics. The assessment of the accessibility of cellulosic materials was performed experimentally through a water vapor sorption method. The kinetics and the thermodynamic parameters of water vapor sorption process were determined, and a correlation between the Flory–Huggins interaction parameters and the crystallinity index was derived. We concluded that the allomorph involving the most accessible crystal surfaces and amorphous regions was Cellulose II. The correlation of the accessibility values with those of the crystallinity index allowed us to evaluate the accessibility of the allomorphic forms of cellulose at different crystallinity indexes. The obtained experimental data allowed us to quantify the accessibility for crystal surfaces and amorphous regions of the different allomorphs in the order Cellulose II (38%) > Cellulose I (24%) > Cellulose III (10%).     

Enzymatic hydrolysis of cellulose I is greatly accelerated via its conversion to the cellulose II hydrate form

Cellulose II hydrate was prepared from microcrystalline cellulose (cellulose I) via its mercerization with 5 N NaOH solution over 1 h at room temperature followed by washing with water. The structure of cellulose II hydrate changed to that of cellulose II after drying. Compared with cellulose II, cellulose II hydrate exhibited a slightly (8.5%) expanded structure only along the direction. The hydrophobic stacking sheets of the cellulose II were conserved in the cellulose II hydrate, and water molecules could be incorporated in the inflated two-chain unit cell of cellulose II hydrate. Enzymatic hydrolysis of cellulose I, cellulose II hydrate, and cellulose II was carried out at 37 °C using solutions comprising a mixture of cellulase and β-glucosidase. The hydrolysis of cellulose II hydrate proceeded much faster than the hydrolysis of the other two substrates, while the saccharification ratio of cellulose II was only slightly higher than that of cellulose I. The alkaline mercerization treatment was also applied to sugarcane bagasse. After its direct mercerization, the cellulose in bagasse was converted from cellulose I to cellulose II hydrate, and then to cellulose II after drying. Similar to in the case of microcrystalline cellulose, the rate of the enzymatic hydrolysis of the mercerized bagasse without drying (cellulose II hydrate) was much faster than the enzymatic hydrolysis of the other two substrates. Thus, the wet forms of cellulose and cellulosic biomass after mercerization, and after hydrolysis with cellulolytic enzymes, afforded superior products with extremely high degradability.     

Effect of a nonionic surfactant on Trichoderma cellulase treatments of regenerated cellulose and cotton yarns

It has been shown that some surfactants affect the hydrolysis of cellulose by cellulase. In this study, the effect of the surfactant Tween 20 on the hydrolysis of different cellulosic fibers was investigated and related to the cellulose fiber structure. It was found that this non-ionic surfactant enhanced the enzymatic saccharification of highly crystalline cellulosics such as Avicel, Tencel and cotton but not of cuprammonium rayon. The enhanced saccha-rification effected by the surfactant is attributed to inhibition of non-productive sorption of the endoglucanase of the cellulose surface which gives greater access to the cellulose chain ends by the exoglucanase. Although all three fibers lost tensile strength as a result of the enzymatic treatment, no further decrease was effected by the presence of the surfactant.     

Synthesis and characterization of microcrystalline cellulose produced from bacterial cellulose

In this study, microcrystalline cellulose (MCC) was prepared from the acid hydrolysis of bacterial cellulose (BC) produced in culture medium of static Acetobacter xylinum. The MCC-BC produced an average particle size between 70 and 90 μm and a degree of polymerization (DP) of 250. The characterization of samples was performed by thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy (SEM). The MCC shows a lower thermal stability than the pristine cellulose, which was expected due to the decrease in the DP during the hydrolysis process. In addition, from X-ray diffractograms, we observed a change in the crystalline structure. The images of SEM for the BC and MCC show clear differences with modifications of BC fiber structure and production of particles with characteristics similar to commercial MCC.     

Enhancing enzymatic hydrolysis of crystalline cellulose and lignocellulose by adding long-chain fatty alcohols

The effects of long-chain fatty alcohols (LFAs) on the enzymatic hydrolysis of crystalline cellulose by two commercial Trichoderma reesei cellulase cocktails (CTec2 and Celluclast 1.5L) were studied. It was found that n-butanol inhibited the enzymatic hydrolysis, but n-octanol, n-decanol and n-dodecanol had strong enhancement on enzymatic hydrolysis of crystalline cellulose in the buffer pH range from 4.0 to 6.0. LFAs can increase the hydrolysis efficiency of crystalline cellulose from 37 to 57 % at Celluclast 1.5L loading of ten filter paper units (FPU)/g glucan. LFAs have similar enhancement on the enzymatic hydrolysis of crystalline cellulose mixed with lignin or xylan. The enhancement of LFAs increased with the decrease of the crystallinity index. LFAs not only enhanced the high-solid enzymatic hydrolysis of lignocellulose, but also improved the rheological properties of high-solid lignocellulosic slurries by decreasing the yield stress and complex viscosity. Meanwhile, LFAs can improve the enzymatic hydrolysis of cellobiose to glucose, especially at low cellulase loading.     

The enzymatic hydrolysis of pretreated agricultural residues and the fermentation of the liberated sugars to ethanol

Agricultural residues were pretreated by steam explosion and the cellulosic component of these substrates were converted to ethanol using a combined enzymatic hydrolysis and fermentation (CHF) process. The enzymatic hydrolysis was carried out using culture filtrates ofTrichoderma harzianum E58 while the liberated sugars were fermented to ethanol byS. cerevisiae. Initially, pretreatment conditions were optimized to ensure that the substrates were readily hydrolyzed and fermented. The agricultural residues were steamed for various times between 30 and 120 s at approximately 240‡C prior to rapid decompression (explosion) in a small masonite-type gun. The various substrates were selectively extracted by water and alkali to see whether the enzymatic hydrolysis and fermentability of the substrates were enhanced. A comparison between the overall conversion of wheat and barley straw was made since these are the two most readily available agricultural residues in Canada. Steam explosion did not affect the hexosan content of the residues, although the pentosan content of the substrates decreased with increasing duration of steaming. The hexosan (cellulose) content of wheat straw was 50.7% of the total substrate while a slightly higher 52.9% cellulose content was detected in the barley straw. Wheat straw was more efficiently hydrolyzed after it had been steamed for 90 s while optimum hydrolysis of the barley straw was detected after 60 s. Steam exploded wheat and barley straw that was subsequently extracted with water was readily hydrolyzed to their component sugars.S. cerevisiae could almost quantitatively convert these sugars to ethanol. This indicated that water washing not only enhanced the enzymatic hydrolysis of the steam exploded substrates, it also removed inhibitory material that restricted the growth of S.cerevisiae. Maximum hydrolysis (78.5%) and ethanol yields (10 mg/mL) were obtained when wheat straw was steamed for 90 s. Slightly lower hydrolysis (76.0%) and ethanol yields (9.5 mg/mL) were obtained with barley straw that had been steamed for 120 s.     

Hydrolysis behavior of various crystalline celluloses treated by cellulase of Tricoderma viride

Cellobiose and glucose are valuable products that can be obtained from enzymatic hydrolysis of cellulose. This study discusses changes in the crystalline form of celluloses to enhance the production of sugars and examines the effect on structural properties during enzymatic hydrolysis. Various crystalline celluloses consisting of group I (cell I, cell III_I, cell IV_I) and group II (cell II, cell III_II, cell IV_II) of similar DPs were prepared as starting materials. The similar DP values allowed a more direct comparison of the hydrolysis yields. The outcomes were analyzed and evaluated based on the residues and supernatants obtained from the treatment. As a result: (1) action of the cellulase of Trichoderma viride decreased both DP and crystallinity, with greater changes in group II celluloses, (2) the polymorphic interconversion process that occurred for cell III_I, cell IV_I, cell III_II and cell IV_II during the treatment was independent of the enzymatic hydrolysis, thus, the hydrolysis behaviors depended on the starting material of the celluloses, and (3) higher sugar production was obtained from cell III_I and group II. Therefore, the hydrolysis behavior of the various crystalline celluloses depended on the particular polymorph of the starting material.     

Assessment of endoglucanase activity by analyzing the degree of cellulose polymerization and high-throughput analysis by near-infrared spectroscopy

We developed a novel and practical assessment technique for endoglucanase (EG) activity by measuring the degree of polymerization (DP) of cellulose from Eucalyptus globulus. This evaluation method demonstrated that EG II from Trichoderma reesei had higher endoglucanase activity than EG I, which has not been detected in conventional experiments using carboxymethyl cellulose as a model substrate. In addition, a high-throughput protocol for DP measurement was developed by using near-infrared spectroscopy combined with a multivariate analysis. Interpreting the regression coefficient, a reciprocal correlation was observed between the relative crystallinity of the cellulosic residue after enzymatic hydrolysis and the DP.     

The structural changes in crystalline cellulose and effects on enzymatic digestibility

The enzymatic hydrolysis of cellulose I achieves almost complete digestion when sufficient enzyme loading as much as 20 mg/g-substrate is applied. However, the yield of digestion reaches the limit when the enzyme dosage is decreased to 2 mg/g-substrate. Therefore, we have performed three pretreatments such as mercerization, dissolution into phosphoric acid and EDA treatment. Transformation into cellulose II hydrate by mercerization and dissolution into phosphoric acid were not sufficient because substrate changed to highly crystalline structure during saccharification. On the other hand, in the case of crystalline conversion of cellulose I to III_I by EDA, almost perfect digestion was achieved even in enzyme loading as small as 0.5 mg/g-substrate, furthermore, hydrolyzed residue was typical cellulose I. The structural analysis of substrate after saccharification provides an insight into relationships between cellulose crystalline property and cellulase toward better enzymatic digestion.     

Differential thermal and thermogravimetric analyses of bound water content in cellulosic substrates and its significance during cellulose hydrolysis by alkaline active fungal cellulases

Various cellulosic substrates were examined for bound water content by differential thermal analysis (DTA) and thermogravimetry (TG). Samples were heated in the range of 30-100 degrees C at a rate of 3 degrees C/min. DTA vaporization curves for different cellulose samples indicated that the bound water (Wf) was vaporized at higher temperature than free water (Wf) at the surface. Weight loss was observed in two stages, corresponding to Wf and Wb in TG curves. The bound water content was dependent on the degree of crystallinity of cellulose. Among different cellulosic substrates, Walseth cellulose showed the highest bound water content, and it also was found to be the least crystalline. The alkaline-active, alkali-stable cellulase was obtained from the alkalotolerant Fusarium sp. The substrate specificity and viscometric characteristics confirmed the enzyme to be an endoglucanase. The Wb content of Walseth cellulose was lowered during the enzymatic hydrolysis. The possible application of bound water analysis in understanding the hydrolysis of cellulosic substrates of different crystallinity is discussed.     

Effect of structural and physico-chemical features of cellulosic substrates on the efficiency of enzymatic hydrolysis

Effects of major physicochemical and structural parameters of cellulose on the rate and degree of its enzymatic hydrolysis were tested with cellulosic materials from various sources. Some different pretreatments were: mechanical (milling), physical (X-ray irradiation), and chemical (cadoxen, H₃PO₄, H₂SO₄, NaOH, Fe²+/H₂O₂). The average size of cellulose particles and its degree of polymerization had little effect on the efficiency of enzymatic hydrolysis. For samples of pure cellulose (cotton linter, microcrystalline cellulose, α-cellulose), increase in the specific surface area accessible to protein molecules and decrease in the crystallinity index accelerated the enzymatic hydrolysis (the correlation coefficients were 0.89 and 0.92, respectively). In the case of lignocellulose (bagasse), a quantitative linear relationship only between specific surface area and reactivity was observed.     

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.     

Characterization of alkali treated flax fibres by means of FT Raman spectroscopy and environmental scanning electron microscopy

Flax fibres grown under well managed conditions were submitted to NaOH chemical treatments, so called Mercerization. The extent of the polymorphic transformation of cellulose I into cellulose II taking place within the crystalline domains of the fibre cellulose was dependent on the alkali concentration. FT Raman spectroscopy turned out to represent an ideal tool for detecting the polymorphic transformation of the cellulosic fine structure of the flax fibres in vivo. In addition to the differences of the FT Raman spectra in the frequency range below 1500 cm(-1), second derivatives of the spectra in the range of the CH stretching vibrations could also be used to distinguish the two polymorphic modifications. The intensity ratio R of the stretching modes v(s)COC and v(as)COC represents a spectral parameter characterising the molecular structure of the flax fibres. As a supplementary tool, Environmental scanning electron microscopy (ESEM) was used to visualize the microstructural fibre properties dependent on the alkali concentrations during the Mercerization.     

Morphological investigation of nanoparticles obtained from combined mechanical shearing,and enzymatic and acid hydrolysis of sisal fibers

The goal of this work was to prepare cellulosic nanoparticles using different processing routes, viz. a combination of mechanical shearing, acid and enzymatic hydrolysis. It was shown that the enzymatic hydrolysis pretreatment of bleached sisal pulp helps the preparation of well individualized rod-like nanocrystals. The morphology of cellulose fibers and nanoparticles was determined by scanning and transmission electron microscopies, respectively. The main outcome of this study indicated the usefulness of the enzymatic treatment for cellulose nanocrystals production. The enzymatic treatment allowed production of a broad range of cellulosic nanoparticles. This investigation proved that the distinction between MFC and whiskers to describe such cellulose nanoparticles is not sufficient. Indeed, it appears essential to indicate the pretreatment performed.     

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-α, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.     

Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, β-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.     

Cellulose hydrolysis – the role of monocomponent cellulases in crystalline cellulose degradation

Changes in the molecular structure of cellulose during hydrolysis with four recombinant -1,4-glycanases from the cellulolytic bacterium Cellulomonas fimi were assessed and compared in an attempt to elucidate the mechanism of crystalline cellulose degradation. It was apparent that the two endoglucanases, Cel6A and Cel5A, degraded sigmacell cellulose differently; Cel5A liberated more soluble sugars (cellobiose and cellotriose) and significantly altered the molecular weight distribution, while Cel6A had a limited effect on the polymer size and liberated primarily cellobiose and glucose. Additionally, both endoglucanases slightly increased the crystallinity of cellulose. In contrast, the cellobiohydrolases, Cel6B and Cel48A, had no effect on cellulose molecular weight and liberated only cellobiose and cellotriose. However, Cel48A was shown to be effective at reducing the crystallinity of the cellulosic substrate, while Cel6B increased the crystallinity index. Synergistic hydrolysis using combinations of the different enzymes showed that, although the cellulose was extensively hydrolysed, the molecular structure of the substrate was similar to the original material. This phenomenon suggests that the actions of individual monocomponent enzymes are offset by the concurrent modification by the complementing enzymes during synergistic hydrolysis.