Use of thermogravimetric analysis to monitor the effects of natural laccase mediators on flax pulp

The effects of 1-hydroxybenzotriazole (HBT) and the natural laccase mediators gallic acid, caffeic acid and p-hydroxybenzoic acid, on the enzymatic bleaching of flax pulp were compared. The treatment was performed under atmospheric air and oxygen pressure, and, for the first time, monitored by thermogravimetric analysis (TGA) for comparison with chemical analysis, FTIR and X-ray diffraction (XRD) spectroscopies. Thermogravimetric data were simulated by applying a nucleation kinetic equation to a combustion model based on four pseudo-components (hemicellulose, amorphous and crystalline cellulose, and lignin). The results thus obtained show that the effects of the natural mediators are similar to those of HBT and lead to increased order in cellulose microfibril surfaces. An increase in pulp crystallinity was additionally exposed by the XRD and FTIR techniques, and a reduction in microfibril equatorial size by the XRD patterns. Simulated DTG curves were used to determine the kinetic parameters for thermal degradation.     

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose

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

The effect of xylanase on lignocellulosic components during the bleaching of wood pulps

HPLC, SEM and XRD techniques have been proposed as methods for ascertaining the changes occurring in polysaccharides (cellulose and xylans) and fibres during the xylanase bleaching processes. TCF and ECF bleached pulps with and without enzyme pretreatment were analysed. The ratio of carbohydrates present in the pulp, observation of changes occurring in the surface of the fibres and the crystallinity and accessibility of the bleached fibres were determinated. These characteristics have been related with pulp properties. Xylan content decreased when pulp was bleached. Xylanase treatment substantially reduced the xylose content present in pulp, measured by HPLC after the hydrolysis method of the sample. Morphological changes in the fibres occurred when the enzymatic treatment was applied. Bleaching increased the crystallinity of the pulp and enzyme pretreatment also affected the crystallinity of cellulose fibres     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Crystallinity of lyophilised carrot cell wall components

The aim of this work was to investigate the effect of removal of cell wall components on the crystallinity of cell walls using X-ray diffraction. Various insoluble cell wall residues were prepared following a sequential extraction of carrot cell wall material. X-ray diffraction patterns were typical of cellulose although there was a possible contribution of pectic polysaccharides to the crystallinity. As more amorphous material was removed to produce a cellulose rich residue, the crystallinity index increased from 12 to 16%, larger than that estimated from cellulose alone. For the last residue treated with 4M KOH, a lower value of crystallinity was found (14%) which resulted from the change of some crystalline domains of cellulose into amorphous regions. Pressing conditions (temperature, water content) have been investigated and did not alter the crystallinity index significantly.     

Structural comparison and enhanced enzymatic hydrolysis of the cellulosic preparation from Populus tomentosa Carr., by different cellulose-soluble solvent systems

This study aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharification. LiOH/urea, LiCl/DMAc, concentrated phosphoric acid, ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and N-methyl-morpholine-N-oxide (NMMO) were selected as the cellulose dissolution agents. Except the cellulosic sample regenerated from LiCl/DMAc system, all the other treated samples exhibited lower cellulose crystallinity and degree of polymerization (DP), and consequently, exhibited a significant enhancement on enzymatic hydrolysis kinetic. Ionic liquid pretreatment offered unique advantages in the hydrolysis rate in the first 10 h, probably due to the extensively structural transformation of cellulose from the crystalline to the amorphous region. Meanwhile, the regenerated cellulose from concentrated phosphoric acid almost completely consisted of cellulose II, and achieved the highest saccharification yield.     

Chlorine-free extraction of cellulose from rice husk and whisker isolation

This work reports the isolation of cellulose whiskers from rice husk (RH) by means of an environmental friendly process for cellulose extraction and bleaching. The multistep process begins with the removal of pectin, cutin, waxes and other extractives from rice husk, then an alkaline treatment for the removal of hemicelluloses and lignin, and a two-step bleaching with hydrogen peroxide/tetra-acetylethylenediamine (TAED), followed by a mixture of acetic and nitric acids, for further delignification of the cellulose pulp. The techniques of infrared absorption spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), modulated differential scanning calorimetry (MDSC) and X-ray diffraction (XRD) showed that the overall process is adequate to obtain cellulose with high purity and crystallinity. This cellulose was submitted to sulfuric acid hydrolysis with the aim to isolate the whiskers. They showed the typical elongated rod-like aspect as revealed by transmission electron microscopy (TEM) and atomic force microscopy (AFM).     

Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation

Sequential regioselective periodate-chlorite oxidation was employed as a new and efficient pretreatment to enhance the nanofibrillation of hardwood cellulose pulp through homogenization. The oxidized celluloses with carboxyl contents ranging from 0.38 to 1.75 mmol/g could nanofibrillate to highly viscous and transparent gels with yields of 100-85% without clogging the homogenizer (one to four passes). On the basis of field-emission scanning electron microscopy images, the nanofibrils obtained were of typical widths of approximately 25 ± 6 nm. All of the nanofibrillar samples maintained their cellulose I crystalline structure according to wide-angle X-ray diffraction results, and the crystallinity index was approximately 40% for all samples.     

Improved accessibility and reactivity of dissolving pulp for the viscose process: pretreatment with monocomponent endoglucanase

A high accessibility is an essential prerequisite for a homogeneous substitution of cellulose material. In this study, chemical and enzymatic pretreatments to increase the accessibility of cellulose materials have been investigated. Dissolving pulp has been treated with a monocomponent endoglucanase. Fock's method, a microscale process similar to the viscose process, showed an increase in cellulose yield. Simultaneously, the viscosity decreased. To clarify whether the increase in reactivity was due solely to the decrease in the degree of polymerization, the dissolving pulp was also subjected to acid hydrolysis. At a given viscosity level, the enzymatic pretreated pulp had a higher reactivity than the pulp subjected to acid hydrolysis. To achieve 100% reactivity, according to Fock, the acid-treated pulp showed a lower molecular weight compared to the enzymatic-treated pulp. A monocomponent endoglucanase can thus be used to increase the reactivity and accessibility of dissolving pulp in the viscose process.     

Pulp properties and their influence on enzymatic degradability

Endoglucanase treatment of pulp for the adjustment of viscosity and the increase in pulp reactivity is a promising step in the concept for the beneficial production of dissolving pulps from paper grade pulps. To promote the commercial applicability of these enzymes, the influence of pulp properties such as carbohydrate composition, pulp type and cellulose morphology on the enzymatic degradability of a pulp was examined. High contents of hemicelluloses and lignin were shown to impair the accessibility of the cellulose to the enzymes. Due to the elevated swelling capacity of cellulose II, conversion of the cellulose morphology from I to II upon alkaline treatments showed a large increasing effect on the cellulose accessibility, and enzymatic degradability. Reactivity measurements of softwood sulfite pulps after enzymatic degradation and acid-catalyzed hydrolysis, respectively, revealed elevated reactivity for the pulp after acid treatment. This is in contrast to effects of enzyme treatments reported for CCE treated kraft pulps.     

Effects of cellulase on the modification of cellulose

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.     

Structural properties of cellulose and cellulase reaction mechanism

The effects of structural properties and their changes during cellulose hydrolysis on the enzymatic hydrolysis rate have been studied from the reaction mechanism point of view. Important findings are the following: (1) The crystallinity index (CrI) of partially crystalline cellulose increases as the hydrolysis reaction proceeds, and a significant slowing down of the reaction rate during the enzymatic hydrolysis is, in large part, attributable to this structural change of cellulose substrate. (2) The crystallinity of completely disordered cellulose, like phosphoric-acid-treated cellulose, does not change significantly, and a relatively high hydrolysis rate is maintained during hydrolysis. (3) The specific surface area (SSA) of partially crystalline cellulose decreases significantly during enzymatic hydrolysis while the change in SSA of regenerated cellulose is found to be negligible. (4) The value of degree of polymerization (DP) of highly ordered crystalline cellulose remains practically constant whereas the change in DP of disordered regenerated cellulose is found to be very significant. (5) Combination of these structural effects as well as cellulase adsorption, product inhibition, and cellulase deactivation all have important influence on the rate of cellulase reaction during cellulose hydrolysis. More experimental evidence for a two-phase model, which is based on degradation of cellulose by simultaneous actions of cellulase complex on the crystalline and amorphous phases, has been obtained. Based on experimental results from this study and other results accumulated, the mode of cellulase action and a possible reaction mechanism are proposed.     

Preparation of cellulose films from solution of bacterial cellulose in NMMO

Bacterial cellulose (BC) was dissolved in N-methylmorpholine N-oxide (NMMO) to prepare regenerated BC films (RBC) with phase inversion. The solubility of BC, supermolecule on structure, morphology, thermal and physical properties of the films were investigated by Fourier transform infrared spectroscopy (FT-IR), solid-state cross polarization/magic angle spinning ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR), wide-angle X-ray diffraction (WAXD), scanning electron microscope (SEM), and thermogravimetric analysis (TGA). The investigation suggested BC was dissolved completely in NMMO. From the C₆ signal shifts to the amorphous area, the crystallinity of materials decreased from 79.20% to 38.17%, and the transformation from cellulose I to II occurred. It was also found that the banded structure of the native materials was replaced by homogeneous and densified sections, so RBC films had better mechanical and barrier properties, and do thermal stability was similar to that of the native BC.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

HNO3/H3PO4–NANO2 mediated oxidation of cellulose — preparation and characterization of bioabsorbable oxidized celluloses in high yields and with different levels of oxidation

The reaction of cellulose with a mixture of HNO₃/H₃PO₄–NaNO₂ (2:1:1.4, v/v/%w) at room temperature for different time intervals has been investigated to produce oxidized cellulose (OC), a biocompatible and bioresorbable polymer. The results revealed an increase in carboxyl content of OC with increasing reaction time, corresponding to about 8.0, 13.4, 17.4 and 18.4% carboxyl content after 12, 24, 36, and 48 h, respectively. The yield of OC ranged between 75 and 81%. The use of different ratios of HNO₃ and H₃PO₄, (11:1, 4:1, 2:1, 1:1, 1:2, and 1:4; v/v), in the reaction had no significant effect on the carboxyl content and yield of the OC products. All products, as produced, were low crystallinity (27–35%) fibrous materials. The length of fibers decreased with increasing reaction time. After ball milling for 24 h, the length of fibers further decreased and products converted into a fine powder consisting of small fibers and aggregated non-fibrous particles. The degrees of polymerization (DP) of the OC products produced after 12, 24, and 48 h of reaction duration were 81, 63, and 53, respectively. After ball milling for 24 h, the corresponding values changed to 57, 51 and 46. However, no significant change in the crystallinity of the products was noted after ball milling. The TGA results showed the OC products to be less thermally stable than cellulose. The degradation temperature appears to decrease with increasing carboxyl content. In conclusion, the results show that the low crystallinity OC products can be successfully prepared in high yields and with different levels of carboxyl content from cellulose by treatment with a mixture of HNO₃/H₃PO₄–NaNO₂.     

Effects of Two-Stage Dilute Acid Pretreatment on the Structure and Composition of Lignin and Cellulose in Loblolly Pine

A standard two-step dilute sulfuric acid pretreatment was performed on Loblolly pine to enhance the overall efficiency of enzymatic deconstruction of woody biomass to monomeric sugars. The structure of milled wood lignin and cellulose isolated from the untreated and acid-treated biomass was studied in detail. Solid-state ¹³C NMR spectroscopy coupled with line shape analyses has been employed to elucidate cellulose crystallinity and ultrastructure. The results indicate an increase in the degree of crystallinity and reduced relative proportion of less ordered cellulose allomorphs following the acid pretreatment. This increase was attributed to a preferential degradation of amorphous cellulose and less ordered crystalline forms during the high temperature pretreatment. Milled wood lignin structural elucidation by quantitative ¹³C and ³¹P NMR reveals an increase in the degree of condensation of lignin due to the pretreatment. The increase in degree of condensation is accompanied by a decrease in β-O-4 linkages which were fragmented and recondensed during the high temperature acid-catalyzed reactions.     

Effect of beating on recycled properties of unbleached eucalyptus cellulose fiber

The effects of beating on recycled properties of eucalyptus cellulose fiber were studied by analyzing the changes of morphological parameters (fiber length and the fines content), physical properties (tensile strength, breaking length and the stretch), WRV, crystal structure of cellulose and pore structure of cellulose fiber. The results showed that beating caused the fine content increase. Tensile strength, breaking length and the stretch increased with the increasing beating time. WRV of the first cycle beaten eucalyptus pulp was increased by 32.1%, compared to the first cycle unbeaten pulp. WRV increased with the increase in beating degree. However, crystallinity of cellulose increased, and then decreased with an increase in beating degree. FTIR spectra showed that there were no drastic changes in the functional groups of the eucalyptus pulp cellulose during beating. Fiber pore size was gradually diverted into macropore with the increase in beating degree, resulting in the mean pore volume increased.     

A new approach to obtain cellulose nanocrystals and ethanol from eucalyptus cellulose pulp via the biochemical pathway

The feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Glucose concentrations from 45 to 125 g/L were obtained after 24 h, with cellulose conversions from 35 to 96%. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol by Saccharomyces cerevisiae, resulting in 62.1 g/L ethanol after 8 h (yield of 95.5%). For all the CCD experimental conditions, the residual solids presented CNF characteristics. Moreover, the use of a new strategy with temperature reduction from 50 to 35°C after 24 h of enzymatic hydrolysis enabled CNC to be obtained after 144 h. The CNC showed a crystallinity index of 83%, length of 260 nm, diameter of 15 nm, and aspect ratio (L/D) of 15. These characteristics are suitable for many applications, such as reinforcement in polymeric materials and other lower volume higher value bio‐based products. The findings indicate the viability of obtaining ethanol and CNC using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1085–1095, 2017     

Facile approach for the dispersion of regenerated cellulose in aqueous system in the form of nanoparticles

This study reports a facile method to disperse cellulose in deionized water, wherein a critical condition of regenerated cellulose is discovered, where it completely disperses up to a maximum of 5 g L(-1) concentration in deionized water with the help of ultrasonication. The dispersed cellulose is characterized by TEM and DLS, the latter among which shows 200 nm hydrodynamic radii of cellulose nanoparticles dispersed in deionized water. FTIR analysis of dispersed cellulose reveals that dispersed cellulose losses its crystallinity during regeneration and dispersion step employed in this study. The dispersed cellulose reported in this study is able to form free-standing, transparent films, which were characterized by SEM, XRD, TGA, EDX, and FTIR spectroscopy and show resistance against dissolution in water. Additionally, the dispersed cellulose is able to undergo at least three times faster enzymatic hydrolysis in comparison to pristine microcrystalline cellulose under similar reaction conditions. The dispersed cellulose reported here could be a better material for reinforcement, preparation of hydrogels, and drug delivery applications under physiological environment.     

Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by N-methylmorpholine-N-oxide

Pretreatment of lignocellulosic biomass by N-methylmorpholine-N-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity after dissolution and precipitation processes. This work focused on assessing several physical changes and their correlations to enzymatic saccharification of pine flour after NMMO pretreatment. Results from microstructure, surface chemical composition, and cellulose accessibility complementarily illustrated the enrichment of cellulose on pine flour surface after NMMO pretreatment. Cellulose accessibility was highly correlated to the overall glucan conversion rate. Changes in crystallinity were correlated to the initial hydrolysis rate but not overall glucan conversion rate. Findings from this work may contribute to lignocellulosic bioenergy from development of novel pretreatment technologies utilizing NMMO.