Behavior of different monocomponent endoglucanases on the accessibility and reactivity of dissolving-grade pulps for viscose process

Three different commercial monocomponent endoglucanases, with and without a cellulose-binding domain (CBD) and differences in their glycosidic hydrolysis mechanisms, were compared with respect to their ability to enhance the accessibility and reactivity of dissolving-grade pulps for viscose production. Hardwood (eucalyptus) and softwood (mixture of Norway spruce and Scots pine) commercial dried and never-dried bleached sulfite dissolving pulps were used for this purpose. The effects of the enzymatic treatments on pulps were studied by reactivity, according to Fock's method, and viscosity measurements, and recording of molecular weight distributions. Among the different assayed enzymes, endoglucanase with a CBD and an inverting hydrolysis mechanism was found to be the most effective in increasing the reactivity of both pulps. Simultaneously, the viscosity decreased, being more marked for softwood dissolving pulp. A narrower molecular weight distribution, with a great reduction in the amount of long-chain cellulose molecules was observed in both pulps, being more pronounced for softwood dissolving pulp. By contrast, endoglucanase without a CBD and a retaining hydrolysis mechanism showed a barley enhancement of the studied properties. The effects of the different endoglucanase treatments were more pronounced when never-dried dissolving pulps were used.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

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.     

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     

Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases

Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested.     

Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

Biological bleaching of chemical pulps

Use of biotechnology in pulp bleaching has attracted considerable attention and achieved interesting results in recent years. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes, xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. The aims of the enzymatic treatment depend on the actual mill conditions and may be related to environmental demands, reduction of chemical costs or maintenance or even improvement of product quality. The use of oxidative enzymes from white-rot fungi, that can directly attack lignin, is a second-generation approach, which could produce larger chemical savings than xylanase but has not yet been developed to the full scale. It is being studied in several laboratories in Canada, Japan, the U.S.A. and Europe. Certain white-rot fungi can delignify kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzyme manganese peroxidase and laccase can also delignify pulps and enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. Development work on laccase and manganese peroxidase continues. This article presents an overview of developments in the application of hemicellulase enzymes, lignin-oxidizing enzymes and white-rot fungi in bleaching of chemical pulps. The basic enzymology involved and the present knowledge of the mechanisms of the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.     

Factors affecting the activation of pulps with laccase

Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new functional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reactions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals.     

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.     

Predicting the most appropriate wood biomass for selected industrial applications: comparison of wood,pulping, and enzymatic treatments using fluorescent-tagged carbohydrate-binding modules

Background

Lignocellulosic biomass will progressively become the main source of carbon for a number of products as the Earth’s oil reservoirs disappear. Technology for conversion of wood fiber into bioproducts (wood biorefining) continues to flourish, and access to reliable methods for monitoring modification of such fibers is becoming an important issue. Recently, we developed a simple, rapid approach for detecting four different types of polymer on the surface of wood fibers. Named fluorescent-tagged carbohydrate-binding module (FTCM), this method is based on the fluorescence signal from carbohydrate-binding modules-based probes designed to recognize specific polymers such as crystalline cellulose, amorphous cellulose, xylan, and mannan.

Results

Here we used FTCM to characterize pulps made from softwood and hardwood that were prepared using Kraft or chemical-thermo-mechanical pulping. Comparison of chemical analysis (NREL protocol) and FTCM revealed that FTCM results were consistent with chemical analysis of the hemicellulose composition of both hardwood and softwood samples. Kraft pulping increased the difference between softwood and hardwood surface mannans, and increased xylan exposure. This suggests that Kraft pulping leads to exposure of xylan after removal of both lignin and mannan. Impact of enzyme cocktails from Trichoderma reesei (Celluclast 1.5L) and from Aspergillus sp. (Carezyme 1000L) was investigated by analysis of hydrolyzed sugars and by FTCM. Both enzymes preparations released cellobiose and glucose from pulps, with the cocktail from Trichoderma being the most efficient. Enzymatic treatments were not as effective at converting chemical-thermomechanical pulps to simple sugars, regardless of wood type. FTCM revealed that amorphous cellulose was the primary target of either enzyme preparation, which resulted in a higher proportion of crystalline cellulose on the surface after enzymatic treatment. FTCM confirmed that enzymes from Aspergillus had little impact on exposed hemicelluloses, but that enzymes from the more aggressive Trichoderma cocktail reduced hemicelluloses at the surface.

Conclusions

Overall, this study indicates that treatment with enzymes from Trichoderma is appropriate for generating crystalline cellulose at fiber surface. Applications such as nanocellulose or composites requiring chemical resistance would benefit from this enzymatic treatment. The milder enzyme mixture from Aspergillus allowed for removal of amorphous cellulose while preserving hemicelluloses at fiber surface, which makes this treatment appropriate for new paper products where surface chemical responsiveness is required.

     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö     

Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.     

Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products

Pulps with residual lignin ranging from 6.4-27.4% (w/w) were prepared from mixed softwoods using a proprietary biorefining technology (the Lignol process) based on aqueous ethanol organosolv extraction. The pulps were evaluated for bioconversion using enzymatic hydrolysis of the cellulose fraction to glucose and subsequent fermentation to ethanol. All pulps were readily hydrolyzed without further delignification. More than 90% of the cellulose in low lignin pulps (< or =18.4% residual lignin) was hydrolyzed to glucose in 48 h using an enzyme loading of 20 filter paper units/g cellulose. Cellulose in a high lignin pulp (27.4% residual lignin) was hydrolyzed to >90% conversion within 48 h using 40 filter paper units/g. The pulps performed well in both sequential and simultaneous saccharification and fermentation trials indicating an absence of metabolic inhibitors. Chemical and physical analyses showed that lignin extracted during organosolv pulping of softwood is a suitable feedstock for production of lignin-based adhesives and other products due to its high purity, low molecular weight, and abundance of reactive groups. Additional co-products may be derived from the hemicellulose sugars and furfural recovered from the water-soluble stream.     

Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator

The ability of two natural phenols to act as mediators of the recombinant Myceliophthora thermophila laccase (MtL) in eucalypt-pulp delignification was investigated. After alkaline peroxide extraction, the properties of the enzymatically-treated pulps improved with respect to the control. The pulp brightness increased (3.1 points) after the enzymatic treatment with MtL alone, but the highest improvements were obtained after the MtL treatment using syringaldehyde (4.7 points) and especially methyl syringate (8.3 points) as mediators. Likewise, a decrease in kappa number up to 2.7 points was obtained after the MtL-methyl syringate treatment, followed by decreases of 1.4 and 0.9 points after the treatments with MtL-syringaldehyde and MtL alone, respectively. On the other hand, removal of the main lipophilic extractives present in eucalypt pulp was observed after the above laccase-mediator treatments. Finally, the doses of both MtL and methyl syringate were reduced, and results compatible with industrial implementation were obtained.     

Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H(2)SO(4) and H(3)PO(4)) such that the cooking liquor pH 相似文献   

Enzymatic Modification of Paper Fibres

The enzymatic mode of action in paper fibre upgrading is still uncertain. In an attempt to clarify how enzymes modify pulp and paper properties, several parameters were analysed in the present work: (i) thermal analysis of the water-solid surface interactions; (ii) fibre cake permeability; (iii) particle size analysis. The results obtained suggest that enzymes modify the interfacial properties of fibres, increasing the water affinity, which in turn change the technical properties of pulp and paper, such as drainability and strength. The modification of paper and pulps following a treatment with cellulose-binding domains further supports this hypothesis.     

Enzymatic Modification of Paper Fibres

The enzymatic mode of action in paper fibre upgrading is still uncertain. In an attempt to clarify how enzymes modify pulp and paper properties, several parameters were analysed in the present work: (i) thermal analysis of the water-solid surface interactions; (ii) fibre cake permeability; (iii) particle size analysis. The results obtained suggest that enzymes modify the interfacial properties of fibres, increasing the water affinity, which in turn change the technical properties of pulp and paper, such as drainability and strength. The modification of paper and pulps following a treatment with cellulose-binding domains further supports this hypothesis.     

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry detecting NOS in healthy and chronically inflamed pulp

The aim of this study was to examine the expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in human dental pulps and determine whether there are changes of the activity in chronically inflamed pulp tissue. Nineteen pulps with clinical diagnosis of chronic pulpitis were collected during endodontic treatment. The healthy controls were obtained from teeth extracted for orthodontic therapy. The clinical diagnosis was confirmed by histological analysis. Healthy pulps showed stratified odontoblasts in peripheral parts, while in central area there was normal connective tissue. Chronically inflamed pulps showed less expressed stratification of odontoblasts and infiltration of lymphocytes, polymorphonuclear leukocytes, plasma cells and mastocytes. NADPH-d granular reactivity was assessed semi quantitatively under the light microscope by a single observer and scored on an intensity scale from negative reaction to very strong reaction. In healthy human pulps, NADPH-d activity was strong to very strong in odontoblastic layer. Endothelial cells and Schwann cells showed strong NADPH-d reactivity, while the other parts of central area were weakly positive. Similar distribution of reactivity was expressed also in chronically inflamed pulp; moderate to strong reaction was observed in stromal area as result of positive reaction in inflammatory cells and endothelial cells of abundant newly formed capillaries.     

Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.