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Bioprocessing of pulp requires lignolytic as well as hemicellulolytic enzymes. The present study is the first report of a cocktail of laccase (L), xylanase (X), and mannanase (M), from a single bacterium for pulp biobleaching. A novel strain Bacillus tequilensis LXM 55 produced thermo-alkali stable L + X + M. On optimization higher enzyme yield (IUml -1 /fold increase) of laccase (396.35/24.16), xylanase (212.95/81.90) and mannanase (153.33/102.90) were achieved in the cocktail. Treatment of pulp with cocktail of enzymes led to 49.35% reduction in kappa number and considerable enhancement in the brightness (11.59%), whiteness (4.11%), and other pulp properties. Most importantly, no mediator system was required for the application of laccase. 40% less chlorine consumption was required to obtain the paper of the same quality as that of pulp treated without enzyme but with 100% chlorine. Therefore, this cocktail of enzymes is highly suitable for pulp biobleaching in the paper mill.

The low solubility of gas molecules in aqueous solutions has limited the power density output of enzymatic biofuel cells. Herein, a single-liquid miniature glucose-O2 fuel cell was constructed by using gas diffusion electrodes, which were prepared by immobilizing glucose oxidase (GOx) or laccase (Lac) modified on a porous structured carbon paper (CP). Due to the fast and direct O2 diffusion from air to the active sites of the immobilized enzyme through the pores of the CP anode/cathode with controlled wettability, the maximum power output densities dramatically increased to 9.64 μW cm-2 at 0.43 V and 53.0 μW cm-2 at 0.45 V for the cell in 5 mM glucose and after exposing the cell to air or O2 atmosphere, respectively. Interestingly, the resulting single-liquid cell could harvest power from human serum operating at a maximum power density of 49.0 μW cm-2 at 0.2 V. The biofuel cell fabricated by the gas diffusion electrodes displayed advantages such as high output power density, low cost and high 'on-chip' integrability and miniaturization, which suggest its great potential for implantable self-powered sensors and for many future applications.

In order to bleach the eucalyptus kraft pulp, two enzyme treatments involving feruloyl esterase and laccase were used in the TCF sequence. Hydroxycinnamic acids, which were released from lignin subunits by the activity of feruloyl esterase, were used as a natural mediator of laccase. The use of sequentially feruloyl esterase and laccase has much higher pulp bleaching effects than the individual enzymes. GthFAE, BmegLac and GthFAE+BmegLac treatments (X) reduced the kappa number of eucalyptus kraft pulps by indicating 9%, 18%, and 30% delignification rates, respectively. Just like in delignification rates, the highest brightness improvement was achieved from the GthFAE+BmegLac combination. The results of the present study indicated that the natural mediators, which are presented in the structure of lignin, could be used as laccase-mediators for pulp bleaching more efficiently and cost-effectively.

Laccase has the enormous potential to be implemented as the multitasking biocatalyst in whole process of paper making. The enzyme can utilize effectively for pulping, delignification of pulps as alone or in the combination with other bleaching enzymes. Laccase has been evaluated for the biografting of pulp fibers, decolorize and stabilize the effluent of the paper mills, biotransformation of high molecular weight (HMW) lignins to lower molecular weight (LMW) aromatic compounds. Further this enzyme has the huge possibilities to apply for deinking of old newsprint (ONP) and pitch removal from varieties of the different pulps. There is no doubt on versatility mode of action of laccases in paper industry, but still there is limited commercialization of this enzyme has been possible, because of laccases has the less redox potential (E0) and needs mediators for the oxidation of non phenolic substrates, production of enzyme is cost intensive at large scale. This review is providing the proper updated information on the state of the art of different applications of laccase in paper industry. It also confer the interpretation to the readers about the areas of extensively studied and the field where there is still much left to be done., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Production of laccase from Ganoderma lucidum RCK 2011 under solid-state fermentation (SSF) conditions was optimized using response surface methodology, resulting in an approximate eightfold increase compared to that in the unoptimized media. Further, the enzyme produced under SSF as whole fermented substrate (in situ SSF laccase) was found to be more stable than the in vitro enzyme (harvested by downstreaming processing of fermented wheat bran). Interestingly, the biobleaching potentials of both in situ and in vitro SSF laccases were comparable, saving 25% chlorine dioxide for achieving similar pulp brightness as obtained in the pulp treated chemically. The reduction in the demand of chlorine dioxide in the pulp bleaching sequence subsequently decreased the levels of adsorbable organic halogen (AOX) in the resulting effluents of the process by 20% compared to the effluents obtained from chemical bleaching sequence. Therefore, direct application of in situ SSF laccase in pulp biobleaching will be environmentally friendly as well as economical and viable for implementation in paper mills.

Laccases are multicopper enzymes present in plants, fungi, bacteria, and insects, which catalyze oxidation reactions together with four electron reduction of oxygen to water. Plant, bacterial, and insect laccases have a polymerizing role in nature, implicated in biosynthesis of lignin, melanin formation, and cuticle hardening, respectively. On the other hand, fungal laccases carry out both polymerizing (melanin synthesis and fruit body formation) as well as depolymerizing roles (lignin degradation). This bifunctionality of fungal laccases can be attributed to the presence of multiple isoforms within the same as well as different genus and species. Interestingly, by manipulating culture conditions, these isoforms with their different induction patterns and unique biochemical characteristics can be expressed or over-expressed for a targeted biotechnological application. Consequently, laccases can be considered as one of the most important biocatalyst which can be exploited for divergent industrial applications viz. paper pulp bleaching, fiber modification, dye decolorization, bioremediation as well as organic synthesis. The present review spotlights the role of fungal laccases in various antagonistic applications, i.e., polymerizing and depolymerizing, and co-relating this dual role with potential industrial significance.

Within this study the mediated detoxification of spent sulphite liquor via laccase from Trametes versicolor was analysed and optimised using an in-situ NMR-spectroscopy method. The enzymatic degradation kinetic was optimised using the degradation rate of aromatic compounds as indirect parameter. Via response surface methodology the impact of the temperature, the pH and the enzyme concentration was analysed and the conditions were optimised focusing on optimal detoxification. The results of the statistical calculation revealed a valid statistical model for the optimal impact on the aromatic degradation with a temperature of 31 °C, a pH of 6 and a laccase concentrations of 179 U g -1 dry matter of spent sulphite liquor. By using these conditions 88.73% of aromatic compounds could be degraded., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A potentially universal approach is presented for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. Bovine serum albumin (BSA) was interlocked in cellulose to form a protein-friendly surface (named BSA-Paper), while also providing COOH and NH 2 groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA-Paper. The second BSA layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA-Paper, and was found to retain over 100 % activity and was 240 times more stable at 25 °C (half life=180 d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The paper and pulp industry is a capital and resource-intensive industry that contributes to ecosystem toxicity and affects human beings., Objective: The study aimed to appraise the potential of xylanases, laccases and manganese peroxidase for the bio-bleaching of paper pulp and to highlight the role of these enzymes as a promising substitute for chlorine-based chemical methods in the bleaching process., Methods: The ligninolytic enzymes including xylanase, laccase and manganese peroxidase isolated from white rot fungi were used for pre-bleaching and bleaching of oven-dried wheat straw pulp., Results: During the sequential enzymatic treatment of oven-dried pulp the brightness was improved and kappa number was reduced by 3.1% and 3.1 points respectively after xylanase treatment, 0.3% and 0.4 points after laccase treatment and 3% and 0.2 points after MnP treatment. During separate treatment of pulp samples with individual enzymes, brightness and kappa number improved by 8% and 3 points respectively after xylanase treatment, by 5% and 1.7 points after laccase treatment and 5% and 1.8 points after treatment with MnP. During subsequent treatment with 4% sodium hypochlorite, the brightness was further improved by 27.9 % for xylanase treated pulp and 29% for the laccase and MnP treated pulp. The xylanase was found most efficient in comparison to laccase and MnP in reduction of kappa number and improvement of brightness., Conclusion: These results clearly indicate the role of laccase, MnP and xylanase from white rot fungi as effective bio-bleaching agents. Therefore, these enzymes can facilitate the bleaching process without threat to environment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.

To reduce pollution and cost of treatment for fresh and recycled paper, co-production of xylanase and laccase was carried out in the same production medium using two compatible species of Bacillus. These co-produced enzymes were used for deinking of old newsprint (ONP) and biobleaching of eucalyptus Kraft pulp. Solid-state co-cultivation of Bacillus sp. and B. halodurans FNP135 was optimized statistically by response surface methodology for the co-production of xylanase (X) and laccase (L). A significant increase in production of xylanase (2.1-fold, 1,685 IU/g) and laccase (2.04-fold, 2,270 nkat/g) was observed under optimized conditions viz. pH (10.5), inoculum size (10 + 10 %) and moisture:substrate ratio (0.8:1). Both the enzymes showed identical temperature and pH optima of 70 °C and 9, respectively, and were used for deinking of ONP pulp and biobleaching of kraft pulp. In case of ONP pulp deinking, the XL treatment increased brightness (11.8 %), freeness (17.8 %), breaking length (34.8 %), burst factor (2.77 %) and tear factor (2.4 %). In case of kraft pulp biobleaching, XL treatment showed a significant increase in brightness (13 %), whiteness (106.15 %) breaking length (49 %), burst factor (6.9 %), tear factor (23 %), and viscosity (11.68 %) and reduction in kappa number (15 %) after alkali extraction and peroxide stage. This enhancement of pulp properties revealed a synergistic effect of xylanase and laccase produced in one setup.

In this work, laccase treatment was employed to enhance nanofiltration process by lignin removal. Results showed that the membrane filterability was increased in terms of deionized water flux and PHL filtration process. On the other hand, the hemicellulosic sugars were negligible affected and can be concentrated to 172 g/L, which was increased about 300% from the original one. The combined laccase-nanofiltration process provides an alternative approach to utilize hemicellulosic sugars of PHL in an environmentally friendly way., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An enzymatic biobleaching sequence (LVAQPO) using a laccase from Trametes villosa in combination with violuric acid (VA) and then followed by a pressurized hydrogen peroxide treatment (PO) was developed and found to give high bleaching properties and meet dissolving pulp requirements: high brightness, low content of hemicellulose, satisfactory pulp reactivity, no significant cellulose degradation manifested by α-cellulose and HPLC, and brightness stability against moist heat ageing. The incorporation of a laccase-mediator system (LMS) to bleach sulphite pulps can be a good alternative to traditional bleaching processes since thermogravimetric analysis (TGA) showed that the laccase treatment prevented the adverse effect of hydrogen peroxide on fibre surface as observed during a conventional hydrogen peroxide bleaching treatment (PO). Although VA exhibited the best results in terms of bleaching properties, the performance of natural mediators, such as p-coumaric acid and syringaldehyde, was discussed in relation to changes in cellulose surface detected by TGA., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A laccase stage can be used as a pre-treatment of a standard chemical bleaching sequence to reduce environmental concerns associated to this process. The importance of each independent variable and its influence on the properties of the bleached pulp have been studied in depth in this work, using an adaptive network-based fuzzy inference system (ANFIS) with four independent variables (laccase, buffer, mediator and oxygen) as input. Eucalyptus globulus kraft pulp was biobleached using a laccase from Pycnoporus sanguineus and a natural mediator (acetosyringone). Later, an alkaline extraction and a hydrogen peroxide treatment were applied. Most biobleaching processes showed a decrease in kappa number and an increase in brightness with no significant impact on the viscosity values, compared with the control. Oxygen was the variable with the smallest influence on the final pulp properties while the laccase and buffer solution showed a significant influence.

Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on laboratory observations only. There are several critical challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)-synthetic or natural, pH, temperature, stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive production at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.

In present study, xylanase and laccase were produced in a cost-effective manner up to 10 kg substrate level and evaluated in elemental chlorine free bleaching of Eucalyptus kraft pulp. Compared to the pulp pre-bleached with xylanase (15%) or laccase (25%) individually, the ClO2 savings were higher with sequential treatment of xylanase followed by laccase (35%) at laboratory scale. The sequential enzyme treatment when applied at pilot scale (50 kg pulp), resulted in improved pulp properties (50% reduced post color number, 15.71% increased tear index) and reduced AOX levels (34%) in bleach effluents. The decreased AOX level in effluents will help to meet AOX discharge limits, while improved pulp properties will be value addition to the paper., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In order to reduce the ecotoxicity of paper mill, four different enzymatic pretreatment strategies were investigated in comparison to conventional chemical based processes. In strategy I, xylanase-aided pretreatment of pulp was carried out, and in strategy II, xylanase and laccase-mediator systems were used sequentially. Moreover, to compare the efficiency of Bacillus stearothermophilus xylanase and Ceriporiopsis subvermispora laccase in the reduction of ecotoxicity and pollution, parallel strategies (III and IV) were implemented using commercial enzymes. Conventional C(D)E(OP)D(1)D(2) (C(D), Cl(2) with ClO2; EOP, H2O2 extraction; D1 and D2, ClO2) and X/XLC(D)E(OP)D(1)D(2) (X, xylanase; L, laccase) sequences were employed with non-enzymatic and enzymatic strategies, respectively. Acute toxicity was determined by the extent of inhibition of bioluminescence of Vibrio fischeri with different dilutions of the effluent. Two-fold increase was observed in EC50 values for strategy I compared to the control process. On the other hand, sequential application of commercial enzymes resulted in higher acute toxicity compared to lab enzymes. In comparison to the control process, strategy II was the most efficient and successfully reduced 60.1 and 25.8% of biological oxygen demand (BOD) and color of effluents, respectively. We report for the first time the comparative analysis of the ecotoxicity of industrial effluents.

Lignin removal is essential for value-added utilization of hemicelluloses and acetic acid present in the prehydrolysis liquor (PHL) of a kraft-based hardwood dissolving pulp production. In this paper, a novel process concept, consisting of laccase-induced lignin polymerization, followed by filtration/flocculation, was developed to enhance the lignin removal. The results showed that the lignin removal increased from 11% to 46-61% at laccase concentration of 1-4 U mL(-1). The GPC results showed that the molecular weight of the lignin from the laccase treated PHL was increased by 160% in comparison with the original one. The subsequent flocculation using singular Poly-DADMAC system or dual polymer system of Poly-DADMAC/CPAM can further remove 10-15% lignin. The concentrations of hemicelluloses and acetic acid were negligibly affected during the laccase treatment, while flocculation caused 12-15% of total sugar loss. Additionally, the process incorporates this new concept into the kraft-based dissolving pulp production process was proposed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bleached cellulose with good end-properties (≈ 90% ISO brightness and 62% cellulose preservation) was obtained by using a totally chlorine-free biobleaching process (TCF). Unbleached sulphite cellulose was treated with Trametes villosa laccase in combination with violuric acid. This enzymatic stage (L) was followed by a chelating stage (Q) and then by a hydrogen peroxide stage reinforced with pressurized oxygen (Po), resulting to an overall LQPo sequence. The use of violuric acid was dictated by the results of a preliminary study, where the bleaching efficiency of various natural (syringaldehyde and p-coumaric acid) and synthetic mediators (violuric acid and 1-hydroxybenzotriazole) were assessed. The outstanding results obtained with laccase-violuric acid system fulfil most of the characteristics of commercial dissolving pulp, totally acceptable for viscose manufacturing or CMC derivatives, with the added advantage that the enzymatic treatment saved 2h of reaction time and about 70% of hydrogen peroxide consumption, relative to a conventional sequence (Po)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hydrophobic properties were conferred to a high-lignin-content Kraft pulp by a laccase-catalysed treatment in the presence of lauryl gallate (LG). The treatment resulted in a two-fold increase in contact angle and conferred water absorption resistance to the pulp. Kappa number was increased, indicating that some phenolic compounds were incorporated in the pulp. A control treatment with LG alone did not affect water absorption, demonstrating that laccase was essential to attain these new properties. The loss of hydrophobicity after an acetone Soxhlet extraction highlighted that adsorbed acetone-soluble compounds played a key role in the properties. GC-FID and HPSEC-UV analysis of the acetone extract indicated the formation of dodecanol and different phenolic oligomers. SEM images showed the treatment-induced changes in the fibre network. Additional experiments with various reaction times and reactant concentrations highlighted the role of LG oxidation products in the introduction of absorption resistance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP)., Methods and Results: The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially., Conclusion: This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

A new approach for the hydrophobization of finished cellulosic substrates based on a previously reported enzymatic technique is proposed. Commercial finished paper was hydrophobized by using laccase from Trametes villosa in combination with lauryl gallate (LG) as hydrophobic compound. The efficiency of the method was increased by the use of a lignosulfonate as a natural dispersant to improve the surface distribution of LG on the paper, raise its hydrophobicity and help preserve the enzyme activity. No similar threefold effect from a single compound for the improvement of enzymatic treatments was previously reported. The influence of processing conditions including the LG dose, treatment time and temperature was also examined, resulting in further increased hydrophobicity. Efficient fiber bonding and chemical functionalization were confirmed by thorough washing and Soxhlet extraction of the paper. As shown here for the first time, enzyme treatments have the potential to improve the surface hydrophobicity of paper-based media., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In thermal printing, bisphenol A (BPA) functions chemically as a developer and reacts with white or colorless dyes in the presence of heat, converting them to a dark color. BPA can transfer readily to skin in small amounts from these papers. Its damage to environment and organisms has caused an extensive concern. In the present study, thermal paper used at the local automated teller machine counters of India were analyzed for the presence of BPA, and the capability of the paper to produce estrogenicity were assessed using a yeast two-hybrid assay experimental system. The study also focused on eliminating the endocrine-disrupting properties with partially purified laccase from newly isolated ascomycete fungi. The results indicate that these papers can produce estrogen hormone-like effect on experimental systems. It should be noted that on a daily basis, tons of such receipts are being dumped in the environment. Estrogenic properties of thermal paper were effectively removed from the reaction mixture within 3 h of incubation with the partially purified enzyme. We propose the utilization of waste thermal paper as a cheap substrate for laccase production for a safer and cleaner environment.

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nine phenol derivatives, p-coumaric acid (PC), vanillin (V), acetovanillone (AV), acetosyringone (AS), syringaldehyde (SA), coniferaldehyde (CLD), ferulic acid (FRC), sinapic acid (SNC), and sinapyl aldehyde (SLD) were assayed as laccase redox mediators in the biobleaching of kenaf and sisal pulps. As a general behaviour, the phenolic mediators increased the kappa number (KN) and reduced the brightness of pulps. In particular, these changes were found to depend in a linear manner on the energy of the highest occupied molecular orbital (E(HOMO)) of the mediators. The phenolic mediator with the lowest E(HOMO) (PC) led to the highest increase of KN and the lowest reduction of brightness. On the contrary, syringyl derivatives (i.e. SA) with high E(HOMO) values caused small KN increases and significant losses of brightness. This behaviour was explained on the basis of a competition between grafting and polymerisation processes. The former basically affects KN, whereas the latter affects pulp brightness., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Modification of the physical properties of old corrugated container (OCC) pulp with laccase or a laccase-mediator (ABTS, HBT, VA) system was investigated under select enzymatic concentrations and reaction times. The optimal conditions for laccase treatment shown to be using a laccase dose of 160U/g o.d. pulp, a treatment time of 20h at 25°C, pH 7 with a pulp consistency of 5%. Results showed that the Lac-HBT treated OCC pulp gave the best strength properties, improving tensile strength by 15.7%. The increase in the carboxyl group content of OCC laccase or Lac-HBT treated pulp led to the increase in the swelling ability and bonding between fibers. Microscope images showed the fiber surface became rougher and more collapsible after Lac-HBT treatment. FT-IR data showed that new carboxylic acid groups were formed during Lac-HBT treatment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A novel laccase producing Basidiomycete Peniophora sp. (NFCCI-2131) was isolated from pulp and paper mill effluent. The optimal temperature and initial pH for laccase production by the isolate in submerged culture were found to be 30 and 4.6° C, respectively. Maltose (20 g l⁻¹) and tryptone (1.0 g l⁻¹) were the most suitable carbon and nitrogen sources for laccase production. Cu²⁺ (1.0 mM) and veratryl alcohol induced maximum laccase production giving 6.6 and 6.07 U/ml laccase activity, respectively. Under optimised culture conditions, 7.6 U/ml activity was obtained, which was 2.4 times higher than that was achieved in basal medium. An evaluation of the delignification efficiency of the crude enzyme in the presence of redox mediators [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and (1-hydroxybenzotriazole)] revealed structural changes in lignin and existence of many active centres for both chemical and biological degradation of lignin following enzymatic treatment.

Introduction of an enzyme and a colour-forming reagent into paper enables the development of an authenticity indicator. The purpose of this work was to study the performance of Trametes versicolor laccase, TvL, and ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt, in various printing substrates when printed with inkjet. The printing substrates included pre-coated mechanical paper additionally coated with PVA, silica and latex. The focus was on the bioanalytical performance and ink penetration. The setting of the printed TvL and ABTS ink was studied visually, with optical and confocal microscopy and with a so-called tape laminating technique. Technical properties of the printing substrates and effect of the surface chemistry were discussed and related to the bioanalytical properties. TvL activity persisted well during the printing. The best colour response was attained using the PVA-coated base paper. The film-forming ability of the PVA was found to be the main contributor to the colour reaction. The uniform, dense and non-porous PVA layer retains the ABTS and TVL molecules on top of the printing substrate. The high local ink concentration on the PVA coating layer combined with the absorptive paper substrate suggests that the PVA film acts as a filtering layer which retains TvL and ABTS molecules in the coating layer but allows most of the ink solvents to penetrate into the paper structure. TvL and ABTS molecules are also trapped in the PVA polymer network due to swelling effect of water. Electrostatic attractions between the PVA and ABTS and TvL molecules do not contribute to the colour reaction., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The environmental impact of enzyme bleaching stages applied to oxygen-delignified eucalypt kraft pulp was assessed via the chemical oxygen demand (COD), color, absorbance spectrum, residual enzyme activity and Microtox toxicity of the effluents from a laccase-HBT (1-hydoxybenzotriazole) treatment. The influence of the laccase and HBT doses, and reaction time, on these effluent properties was also examined. The laccase dose was found to be the individual variable most strongly affecting COD, whereas the oxidized form of HBT was the main source of increased color and toxicity in the effluents. Moreover, it inactivated the enzyme. Oxidation of the mediator was very fast and essentially dependent on the laccase dose. Using the laccase-mediator treatment after a xylanase stage improved pulp properties without affecting effluent properties. This result holds great promise with a view to the industrial implementation of biobleaching sequences involving the two enzymes in the future., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Laccase, through its versatile mode of action, has the potential to revolutionize the pulping and paper making industry. It not only plays a role in the delignification and brightening of the pulp but has also been described for the removal of the lipophilic extractives responsible for pitch deposition from both wood and nonwood paper pulps. Laccases are capable of improving physical, chemical, as well as mechanical properties of pulp either by forming reactive radicals with lignin or by functionalizing lignocellulosic fibers. Laccases can also target the colored and toxic compounds released as effluents from various industries and render them nontoxic through its polymerization and depolymerization reactions. This article reviews the use of both fungal and bacterial laccases in improving pulp properties and bioremediation of pulp and paper mill effluents., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Functionalization of sisal specialty pulp fibers by laccase-catalysed grafting of ferulic acid (FRC) was investigated. To this end, the extent of phenol coupling to fibers under different reaction conditions (laccase and FRC rates, and time) was evaluated in terms of pulp properties including kappa number (expressed as the combined contributions of lignin and hexenuronic acids), brightness, Klason lignin and surface anionic charge after Soxhlet extraction of acetone-treated pulp. The specific treatment resulting in the highest degree of grafting was then used in a comparative study of the effects of applying the laccase-FRC system to refined and unrefined pulp with a view to confirming whether the increased surface area obtained by effect of fibrillation would lead to enhanced grafting. Based on the results, refining the pulp prior to the enzyme treatment resulted in increased grafting which in turn led to handsheets with improved strength-related properties (particularly wet tensile strength) relative to control samples., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 3h, 3UA/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 Na(2)S 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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pulp and paper mills generate pollutants associated to their effluents depending upon the type of process, type of the wood materials, process technology applied, management practices, internal recirculation of the effluent for recovery, the amount of water used in the industrial process and type of secondary treatment. This study is the first that reports a simultaneous evaluation of the effects of tertiary treatments by fungi (Rhizopus oryzae and Pleurotus sajor caju), by enzyme (laccase) and by an oxidation process (photo-Fenton) on individual phenols (vanillin, guaiacol, phloroglucinol, vanillic acid and syringic acid) of a Eucalyptus globulus bleached kraft pulp and paper mill final effluent after secondary treatment (BKPME). The tertiary treatments were applied on BKPME samples and in BKPME samples supplemented with extra concentration of each phenol. Tertiary treatments by Rhizopus oryzae and photo-Fenton oxidation were able of complete removal (100%) of phenols on BKPME samples whereas P. sajor caju and laccase were able of 60-85% removal. On BKPME samples with added concentration of each phenol, photo-Fenton was the only treatment capable of total phenols removal (100%), which suggests a great potential for its application.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aim of this work was to develop an innovative method for the internal sizing of paper by use of laccase and hydrophobic compounds. Nine different products containing hydrophobic moieties were tested in combination with laccase derived from Trametes villosa on Eucalyptus globulus kraft pulp in order to assess their internal sizing capability. The strongest internal sizing effect was obtained with lauryl gallate (LG). Heat treatment of the handsheets was found to increase the resistance to water absorption of internally sized samples significantly. Tests were conducted under variable operating conditions, including enzyme and reactant doses and treatment time. In addition to altering the water absorption rate, internal sizing with the laccase-LG treatments was found to affect the mechanical and optical properties of the handsheets. As shown in this work, treatments based on laccase and a hydrophobic compound (particularly lauryl gallate), can provide a new, effective biotechnological method for the internal sizing of paper., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Application of a laccase mediator system (an L stage) to TCF and ECF bleached pulp from Eucalyptus globulus with low residual lignin content (KN(lig)1.0) provides useful information about its effects on hexenuronic acids, functional groups (carboxyl and carbonyl) and electrokinetic properties such as zeta potential and surface charge. The use of laccase from Trametes villosa in combination with the mediator 1-hydroxybenzotriazole (HBT) was found to oxidize cellulose to carbonyl groups and reduce the amount of carboxyl groups present in TCF pulp by effect of its partially removing hexenuronic acids from it. This result may open up new prospects for improving brightness stability in pulp. In addition, the laccase mediator system modifies the surface charge and zeta potential in the fibre suspension for the removal of ionizables groups in TCF pulp. This result has no adverse effect on the pulp refining efficiency. L treatment requires less mechanical energy than conventionally refined pulp to obtain an optimal tensile and tear index in handsheets. This behavior may be attributable to the modification of the electrokinetic properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Modification of properties of old newspaper (ONP) deinked pulp with laccase and histidine was investigated. It was found that the optimum conditions for laccase-histidine treatment were: the concentration of laccase 0.9 U/g dry pulp, the concentration of histidine 1% relative to the dry pulp, room temperature, reaction time 1.5h, pH 7, the pulp consistency 5% and O(2) atmosphere. The results also showed that, in the optimum conditions, compared to the control pulp, the wet tensile strength, the carboxyl group content and water retention value of ONP treated with laccase and histidine were increased by 55.1%, 39.1% and 45.7%, respectively. Moreover, environmental scanning microscope images showed that more collapse and more fibrillation were observed on the laccase-histidine-treated fiber surface than the control samples, which led to form better bonding between fibers in handsheets resulting in the increase of the paper strength of laccase-histidine-treated ONP pulp., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A new biobleaching sequence, with two enzymatic stages based on the application of laccase-mediator systems, was tested (L(1)EL(2)QPo) in order to increase the effectiveness of enzyme delignification on eucalypt kraft pulp. Different synthetic -1-hydroxybenzotriazole (HBT) and violuric acid (VA) - and natural - syringaldehyde (SyAl) - mediators were used in the laccase stages and the biobleached pulp were compared in terms of chemical, optical and physico-mechanical properties. The pulp bleached with HBT or VA showed similar delignification (64.1% and 65.9% respectively) and optical properties (86.4% and 86.1% ISO brightness respectively) than an industrial TCF pulp (68.3% delignification and 84.8% ISO brightness). SyAl improved these properties in a lower extent (56.71% delignification and 80.52% ISO brightness). Regarding physico-mechanical properties of pulp, the biobleaching sequence had no a negative effect, even some slight improvements were observed in very specific cases., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The great amount of pollutants released from kraft pulp processes, mainly from cooking and bleaching stages, is one of the most relevant environmental problems in this type of industry. New bleaching sequences are being studied based on the use of oxidative enzymes from fungal cultures. In this study, the bleaching systems consisting of Laccase and different mediators such as 1-hydroxybenzotriazole, violuric acid, syringaldehyde and methyl syringate in the bleaching sequence of Eucalyptus globulus kraft pulp were applied. The main objective of this study is to evaluate the aerobic and anaerobic biodegradability and toxicity to Vibrium fischeri of generated L-stage and total bleaching sequence effluents. The highest levels of aerobic and anaerobic degradation of the generated effluents were achieved for treatments with laccase plus violuric acid, with 80% of aerobic degradation and 68% of anaerobic biodegradation. V. fischeri toxicity was remarkably reduced for all the effluents after aerobic degradation., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The efficiency of advanced oxidation processes (AOPs), enzymatic treatment and combined enzymatic/AOP sequences for the colour remediation of soda and chemimechanical pulp and paper mill effluent was investigated. The results indicated that under all circumstances, the AOP using ultraviolet irradiation (photo-Fenton) was more efficient in the degradation of effluent components in comparison with the dark reaction. It was found that both versatile peroxidase (VP) from Bjerkandera adusta and laccase from Trametes versicolor, as pure enzymes, decolorize the deep brown effluent to a clear light-yellow solution. In addition, it was found that in the laccase treatment, the decolorization rates of both effluents were enhanced in the presence of 2, 2'-azinobis (3-ethylbenzthiazoline-6-sulfonate), while in the case of VP, Mn(+2) decreased the efficiency of the decolorization treatment. The concomitant use of enzymes and AOPs imposes a considerable effect on the colour remediation of effluent samples.

High-quality flax pulp was bleached in a short totally-chlorine-free (TCF) sequence. The LP biobleaching sequence includes an enzyme treatment with laccase in the presence of HBT as mediator (L stage) and a hydrogen peroxide stage (P stage). The operating conditions for the laccase HBT system were optimized by using a sequential statistical plan involving four variables, the influence of which on the properties of the pulp after the P stage was examined over enclosed operation conditions regarding a future industrial application. Mathematical models accurately predicting both pulp properties in terms of the previous four variables. This biobleaching sequence allows obtaining an increase in ISO brightness of 40% and a delignification of 80%. Process variables optimization in flax pulp biobleaching allows establishing the application conditions most appropriated to obtain certain pulp properties and that turn out to be easily adaptable to existing industrial processes. As a novelty, toxicity measurements were determined in L stage effluents. The results of toxicity show that it is possible to apply this LP sequence without having a negative environmental impact.

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.

Pycnoporus cinnabarinus laccase was fused to the C-terminal linker and carbohydrate binding module (CBM) of Aspergillus niger cellobiohydrolase B (CBHB). The chimeric enzyme of molecular mass 100 kDa was successfully produced in A. niger. Laccase-CBM was further purified to determine its main biochemical properties. The Michaelis-Menten constant and pH activity profile were not modified, but the chimeric enzyme was less thermostable than either the P. cinnabarinus laccase or the recombinant laccase produced in the same strain. Laccase-CBM was able to bind to a cellulosic substrate and, to a greater extent, to softwood kraft pulp. Binding to the pulp was shown to be mainly time and temperature-dependent. Laccase-CBM was further investigated for its softwood kraft pulp biobleaching potential and compared with the P. cinnabarinus laccase. Addition of a CBM was shown to greatly improve the delignification capabilities of the laccase in the presence of 1-hydroxybenzotriazole (HBT). In addition, ClO(2) reduction using 5 U of chimeric enzyme per gram of pulp was almost double than that observed using 20 U of P. cinnabarinus laccase per gram of pulp. We demonstrated that conferring a carbohydrate binding capability to the laccase could significantly enhance its biobleaching properties.