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.     

Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp

The natural phenolic compounds syringaldehyde and vanillin were compared to the synthetic mediators 1-hydroxybenzotriazole, violuric acid and promazine in terms of boosting efficiency in a laccase-assisted biobleaching of eucalyptus kraft pulp. Violuric acid and 1-hydroxybenzotriazole revealed to be the most effective mediators of the bioprocess. Nevertheless, laccase-syringaldehyde system also improved the final pulp properties (28% delignification and 63.5% ISO brightness) compared to the process without mediator (23% and 61.5% respectively), in addition to insignificant denaturation effect over laccase. The efficiency of the biobleaching process was further related to changes in non-conventionally used optical and chromatic parameters of pulp, such as (L( *)), chroma (C( *)) and dye removal index (DRI) showing good correlation. Adverse coupling reactions of the natural phenolic mediators on pulp lignin were predicted by electrochemical studies, demonstrating the complexity of the laccase-mediator reaction on pulp.     

Application of laccase-natural mediator systems to sisal pulp: An effective approach to biobleaching or functionalizing pulp fibres?

The effects of laccase-natural mediator systems (LMS) on sisal pulp and their potential for either biobleaching or functionalizing (via radical-coupling) its fibres were investigated. The enzyme treatment (L stage) was followed by extraction with hydrogen peroxide in order to determine whether observable effects could be enhanced by removing LMS-modified lignin. Four different plant phenols [viz. the p-hydroxycinnamic compounds sinapic acid (SNC), ferulic acid (FRC), coniferyl aldehyde (CLD) and sinapyl aldehyde (SLD)] were used as laccase redox mediators and their effects on pulp and effluents compared with those of the synthetic compound 1-hydroxybenzotriazole (HBT). During the L stage performed with HBT, laccase underwent a loss of 99% and 78% of the initial activity, in the absence and presence of pulp, respectively. With natural mediators inactivation was markedly reduced, being the residual activity between 65% and 100% of the initial one, in the presence of pulp. The pulp was found to protect the enzyme against inactivation: the activity was only reduced by 45% in its presence. Under the operating conditions used the natural mediators proved less efficient than HBT in facilitating pulp bleaching; rather, they tended to bind to pulp fibres. This effect could be used to functionalize fibres in order to improve intrinsic properties of pulp or introducing novel ones (e.g. antimicrobial, antioxidant, optical properties, etc.). This paper shows for the first time the application of laccase-mediator systems to sisal pulp.     

Laccase for biobleaching of eucalypt kraft pulp by means of a modified industrial bleaching sequence

Biobleaching of kraft pulp is a possible application of laccase, but it has not been described in detail for complete industrial bleaching sequences yet. Therefore, in this work, the biobleaching of Eucalyptus globulus kraft pulp was performed using a modified industrial totally chlorine‐free sequence. The modification consisted in the substitution of an enzymatic delignification stage, based on the application of laccase from Trametes villosa, for the first alkaline extraction one. The enzymatic stage was performed with several synthetic and natural mediators, namely 1‐hydroxybenzotriazole (HBT), violuric acid (VA), methyl syringate, and syringaldehyde. Several pulp properties were analyzed after each stage of the bleaching process—kappa number, ISO brightness, viscosity, and optical properties of CIEL*a*b* system. The new biobleaching sequence improved the pulp properties, in comparison to the conventional bleaching sequence, if HBT or VA was used as mediators. VA was selected as the best mediator of those tested and the effect of its concentration in the enzymatic stage was subsequently studied. Reducing the initial concentration by 30%, the same pulp quality was obtained, but if the reduction attained 60%, an important decrease in pulp integrity was detected. The modified bleaching sequence could improve the bleached pulp properties (kappa number 10%, ISO brightness 1%, and viscosity 5%) in comparison to the mill sequence. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (<50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.     

Biobleaching of Eucalyptus globulus kraft pulps: comparison between pulps obtained from exploded and non-exploded chips

The aim of this work was to evaluate the response to biobleaching of steam exploded kraft pulps and to compare the results with the controls. For this end, a laccase-mediator treatment using commercial laccase (Novozyme 51003) and a natural mediator (acetosyringone) were assayed, followed by alkaline extraction and hydrogen peroxide stages.Our approach resulted in exploded biobleached pulps with lower kappa number and improved optical properties compared to controls, even after subjecting pulps to accelerated ageing. Additionally, use of hydrogen peroxide was reduced. The LMS (laccase-mediator system) had a smaller impact on the properties of the bleached pulps and on hydrogen peroxide consumption than the steam explosion process did.     

Reutilization of effluents from laccase-mediator treatments of kraft pulp for biobleaching

Several effluents from laccase-mediator treatments of kraft pulp were recovered and subsequently reused with fresh pulp in order to simulate recirculation of effluents during biobleaching. The effluents were used as a new bleaching stage without any modification except enzyme addition. Pulp treated with effluents were afterwards chemically bleached by using the simple sequence LQPo, where L represents the treatment with effluent and laccase addition, Q is a chelating stage and Po is an alkaline peroxide stage. This system showed a promising potential on delignification, with kappa number ranging from 5.5 to 6.6 after LQPo sequence, depending on the type of effluent employed in L stage. Improvements on pulp brightness were also reported compared with control experiment.     

Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system

The use of enzymes has been suggested as an environmentally friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of cellulases/hemicellulases versus the laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens, Novozymes) and a commercial laccase (NS51002 from Trametes villosa, Novozymes), the latter in the presence of synthetic or natural (lignin-related) mediators, were evaluated. The enzymatic treatments were studied at the laboratory scale using a standard chemical deinking sequence consisting of a pulping stage; an alkaline stage using NaOH, sodium silicate and fatty acid soap; and a bleaching stage using hydrogen peroxide. The handsheets were then prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3–4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. Regarding the laccase-mediator system, alone or in combination with carbohydrate hydrolases, it was ineffective in deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by the laccase-mediator system was displayed when secondary fibers from printed cardboard were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations.     

Combination of steam explosion and laccase-mediator treatments prior to Eucalyptus globulus kraft pulping

The effect of a pretreatment consisting of steam explosion (SE) followed by a laccase mediator system (LMS) stage on Eucalyptus globulus kraft pulping has been evaluated and compared with fungal pretreatments. Pretreatment with SE and LMS was more efficient than pretreatments using Pycnoporus sanguineus and Trametes sp. I-62. Steam explosion not only improved the enzyme penetration into the wood chips and shortened the pulping process by 60%, but also extracted around 50% of the hemicelluloses which could be converted into value-added products. The optimal conditions for the LMS treatment were 3 h, 3 UA/g and 40 °C. Compared to SE, the SE/LMS treatment yielded an increase in delignification of 13.9% without affecting pulp properties, provided a similar screened kraft yield, and reduced consumption of chemical reagents Na₂S and NaOH by 11.5% and 6.3%, respectively. Therefore, SE/LMS is a promising pretreatment for converting the pulp mill into a forest bio-refinery.     

Laccase-mediator biobleaching applied to a direct yellow dyed paper

A laccase-mediator system (LMS) for biobleaching was applied to a bleached chemical pulp dyed with stilbene dye Direct Yellow 11. Of mediators tested, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was found to be more effective than either violuric acid (VA) or N-hydroxybenzotriazole (HBT), which had been shown to be superior to ABTS when bleaching virgin chemical pulp. The laccase-ABTS system removed more than 60% of the color.     

Laccase-HBT bleaching of eucalyptus kraft pulp: influence of the operating conditions

Different operating conditions (viz. pulp consistency, oxygen pressure and treatment time) in the biobleaching of eucalyptus kraft pulp with the laccase-HBT system was tested in order to describe their effect and normalize a biobleaching protocol. A high O(2) pressure (0.6MPa) was found to result in improved laccase-assisted delignification of the pulp. Also, a high pulp consistency (10%) and a short treatment time (2h) proved the best choices with a view to obtaining good pulp properties (kappa number and ISO brightness) under essentially mild conditions. The laccase-HBT treatment was found to result in slight delignification (in the form of a 20-27% decrease in kappa number); however, an alkaline extraction stage raised delignification to 41-45%, a much higher level than those obtained in the control tests (16-23%). Also, the use of hydrogen peroxide in the extraction stage resulted in improved brightness (14-19%), but in scarcely improved delignification (4-7%). Treating the pulp with the laccase-HBT system reduced the amount of hydrogen peroxide required for subsequent alkaline bleaching by a factor of 3-4 relative to control tests.     

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Laccase-induced HBT-grafting to milled beech wood reduces unspecific protein adsorption

Lignocellulosic biomass is a ubiquitous and renewable feedstock for the production of platform chemicals and biofuels. Typically, this recalcitrant biomass is pretreated by physico-chemical techniques causing disintegration and delignification. An additional treatment with laccase-mediator-systems (LMS) has been found to further improve the subsequent enzymatic cellulose hydrolysis. The aim of this study was to investigate the impact of different LMS on the glucose yield of a subsequent hydrolysis of treated beech wood and to elucidate the underlying effect of LMS treatment. The mediators 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1-hydroxybenzotriazol (HBT) and syringaldehyde were evaluated, but an enhancing effect of LMS treatment on beech wood hydrolysis was only found for HBT. In mass spectrometry analysis of the acid hydrolysate of LMS-treated samples, the mediator HBT could be found in the lignin samples, suggesting a grafting reaction. The fluorescent protein mCherry was used as a reporter for unspecific protein adsorption to biomass samples. LMS treatment with HBT reduced the unspecific adsorption of mCherry to raw beech wood by about 50%, suggesting that the HBT grafting to beech wood lignin decreased the unproductive cellulase binding. In summary, the reduction of unspecific protein adsorption by biomass surface modification with laccase-HBT treatment is proposed to be the underlying mechanism for increased cellulose conversion.     

New polyoxometalate–laccase integrated system for kraft pulp delignification

New polyoxometalate–laccase integrated system (PLIDS) employing polyoxometalate [SiW₁₁V^VO₄₀]⁵⁻ and laccase of Trametes versicolor for the continuous delignification of eucalypt kraft pulp has been developed. Pulp was delignified in a batch reactor containing catalytic amounts of [SiW₁₁V^VO₄₀]⁵⁻ at about 90 °C under atmospheric pressure. Re-oxidation of reduced polyoxometalate (POM) with laccase was carried out at 45 °C in a separate aerated bioreactor coupled with an ultrafiltration tubular ceramic membrane. This allowed the separation of laccase from re-oxidized POM, which was supplied in turn continuously to the delignification reactor.Proposed PLIDS allowed sustainable pulp delignification with minimal degradation of polysaccharides. The implementation of PLIDS, instead the fist chlorine dioxide stage (D) in conventional DEDED bleaching sequence, showed almost 60% of chlorine dioxide savings with strength properties of the bleached pulp (90% ISO) similar to those obtained after the conventional bleaching.     

Fiber surface characterization of old newsprint pulp deinked by combining hemicellulase with laccase-mediator system

Deinking of old newsprint (ONP) by combining hemicellulase with laccase-mediator system (LMS) was investigated, and surface chemical composition and fiber morphology changes during the deinking process were studied by electron spectroscopy for chemical analysis (ESCA), contact angle (CA), attenuated total reflectance fourier transform infrared spectrometry (ATR-FTIR), fiber quality analyzer (FQA), and environmental scanning electronic microscopy (ESEM). Results showed that, compared to the pulp deinked with hemicellulase or LMS individually, effective residual ink concentration (ERIC) was lower for the hemicellulase/LMS-deinked pulp. This indicated that there is a synergistic deinking effect between hemicellulase and LMS. It was found that O/C ratio of the fiber surface increased and the surface coverage of lignin decreased during the hemicellulase/LMS deinking process. The contact angle of the hemicellulase/LMS-deinked pulp was lower than that of pulps deinked with each individual enzyme. ESEM observations showed that more fibrils appeared on the fiber surface due to synergistic treatment.     

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Influence of operation conditions on laccase-mediator removal of sterols from eucalypt pulp

The way how sterols, the main lipophilic compounds present in eucalypt kraft pulp, are eliminated by an enzymatic stage using the laccase-mediator system was evaluated. With this purpose laccase-mediator stage (L) was applied on an Eucalyptus globulus pulp under different operation conditions following a three-variable (laccase dose, mediator dose and reaction time) sequential statistical plan, to optimise the removal of sterols. The decrease in pulp sterol content during the enzymatic treatment was related to the decrease in kappa number and to brightness increase, as well as with the increase in some oxidation products of sitosterol (namely 7-oxositosterol and stigmasta-3,5-dien-7-one). The increase in reaction time from 1 to 5 h strongly reduced the sterol content, while no more sterols were eliminated during the 5–7 h period. Increasing the laccase dose from 1 to 20 U g⁻¹ of pulp produced a high reduction in pulp sterols, whereas the increase in mediator (1-hydroxybenzotriazole) dose (from 0.5 to 2.5% of pulp weight) had only a slight influence in removing sterols. Therefore, at 16 U g⁻¹ laccase dose, 0.5% mediator dose, 4 h of reaction, practically all the sterols were removed. Finally, it was demonstrated that sterols were more sensitive to a L stage (practically 100% of sterols were eliminated) than to a chlorine dioxide stage (54% of sterols eliminated).     

Laccase mediator systems for eco-friendly production of medium-density fiberboard (MDF) on a pilot scale: physicochemical analysis of the reaction mechanism

Increasing prices of petrochemical resins and possible harmful formaldehyde emissions from conventionally produced wood composites have resulted in increased interest in enzymatic binder systems as environmentally friendly alternatives for gluing lignocellulosic products. In this study, laccase mediator systems (LMSs) were used to activate lignin on wood fiber surfaces in the pilot-scale production of medium-density fiberboard (MDF) using a dry process. Three different mediators were applied: 4-hydroxybenzoic acid (HBA), 1-hydroxybenzotriazole (HBT), and acetosyringone (AS) of which HBA performed best. The mechanical properties of the manufactured boards produced with thermomechanical pulp (TMP) fibers, laccase, and HBA fulfilled all required European standards for wood-based panels. Oxygen consumption rates of the different LMSs and (13)C NMR spectroscopy results for treated TMP fibers were obtained for qualitative and quantitative analysis of lignin activation. The results show that reactions were most effective within the first 30 min of incubation. Oxygen consumption was fastest and highest for the LMS using HBA. (13)C NMR spectroscopy indicated the highest decrease of aromatic groups in the wood fiber lignin with this LMS. The data correlated well with the quality of the MDF. The required enzymatic reaction times allowed direct integration of the LMS into standard MDF production techniques. The results indicate that application of LMSs has a high potential for environmentally friendly MDF production.     

Transformation parameters enhancing T-DNA expression in kenaf (Hibiscus cannabinus)

Kenaf(Hibiscus cannabinus)is a fast growing annual with tremendous potential as a source of fiber for ropes, textiles and paper. Kenaf is an environmentally friendly crop; however, commercial production of kenaf is hindered by weed competition at the seedling stage. Herbicide resistant kenaf cultivars would reduce seedling weed competion and make growing kenaf more profitable. Factors that are important in establishing a transformation system for kenaf were examined. The influence of Agrobacterium strain, temperature, host tissue wounding, acetosyringone, virG/virE genes and host cell division on T-DNA expression in the kenaf shoot apex were investigated. Three Agrobacterium strains were tested, and A. tumefaciens LBA4404 significantly (α=0.05) yielded a high number of shoots surviving on selection medium; no shoots survived with EHA101S or Z707S. There was no significant difference (α=0.05) in transient T-DNA expression between 28 °C and 25 °C; however, shoots did not survive 16 °C or 19 °C co-cultivation temperatures. Shoot apex survival was increased significantly (α=0.05) when virulence genes and a cytokinin, TDZ, were combined. Sonicated shoots showed an increase in transient expression and shoot survival. Optimal conditions for shoot apex T-DNA transfer and expression were sonication for 5 s, co-cultivation with LBA4404 containingvirG/virEat room temperature, and 200 μmol/L acetosyringone.     

Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.