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In present research, a potent fungal strain was isolated from paper mill effluent (black liquor) in order to investigate its potential for the biodegradation of lignin. Two step strategy was used to screen most efficient fungal strain having ability to growin MSM-black liquor medium and to degrade alkali lignin.The results of initial screening indicated that the strain M-2 produced comparatively higher ligninolytic zone on MSN agar plates supplemented with black liquor (BL) and alkali ligninase compared to the other isolates.The results of 18S rRNA gene sequencing revealed that strain M-2 showed ≥ 99% sequence homology with Dipodasceus australiansis.The process for the biodegradation of lignin was optimized using Taguchi Orthogonal Array design. Under optimized conditions of pH 9, 40 °C and 4% inoculum, a maximum of 89% lignin was degraded with 41% color reduction after 8 days of incubation period by Dipodasceus australiansis M-2. The pH and temperature were found to be significant terms with the p-values of 0.002 and 0.001 respectively. The laccase activity of the Dipodascus australiensis was found to be maximum of 1.511 U/mL. The HPLC analysis of lignin biodegradation indicated sharp transformation of peaks as compared to the control. Our results suggested that the strain Dipodascus australiensis M-2 possess excellent lignin degradation and color reduction capability and can be applied in waste treatment systems for pulp and paper mill effluent. In present work we are reporting first hand information regarding biodegradation of lignin by a potent strain of Dipodascus australiensis and statistical optimization of the bioprocess.

Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40 °C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents. HIGHLIGHTS Bacillus altitudinis SL7 isolated from effluent could efficiently degrade lignin under alkaline conditions.; Degradation efficiency was determined by analyzing lignin content, color reduction, and ligninolytic enzyme activity.; Extracellular laccase from B. altitudinis SL7 can play a significant role in the depolymerization of lignin.; Various low-molecular-weight lignin degradation products were determined through GC-MS.

The pulp and paper industry discharges massive amount of wastewater containing hazardous organochlorine compounds released during different processing stages. Therefore, some cost-effective and nonpolluting practices such as enzymatic treatments are required for the potential mitigation of effluents released in the environment. Various xylanolytic enzymes such as xylanases, laccases, cellulases and hemicellulases are used to hydrolyse raw materials in the paper manufacturing industry. These enzymes are used either individually or in combination, which has the efficient potential to be considered for bio-deinking and bio-bleaching components. They are highly dynamic, renewable, and high in specificity for enhancing paper quality. The xylanase act on the xylan and cellulases act on the cellulose fibers, and thus increase the bleaching efficacy of paper. Similarly, hemicellulase enzyme like endo-xylanases, arabinofuranosidase and β-D-xylosidases have been described as functional properties towards the biodegradation of biomass. In contrast, laccase enzymes act as multi-copper oxidoreductases, bleaching the paper by the oxidation and reduction process. Laccases possess low redox potential compared to other enzymes, which need some redox mediators to catalyze. The enzymatic process can be affected by various factors such as pH, temperature, metal ions, incubation periods, etc. These factors can either increase or decrease the efficiency of the enzymes. This review draws attention to the xylanolytic enzyme-based advanced technologies for pulp bleaching in the paper industry.

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.

This paper establishes a membraneless, co-laminar flow-based approach to develop a cost-effective microfluidic paper-based analytical device for enzymatic biofuel cell ( $\mu $ PAD-EBFC). The developed $\mu $ PAD-EBFC supporting the self-capillary fluid transport action consists of Y-shaped paper microchannel with the fuel (glucose) and oxidant (O2) streaming in parallel over carbon nanotube-based bucky paper electrodes modified with biocompatible electrocatalytic enzymes, such as glucose oxidase and laccase without any additional redox cofactor. The electrochemical performance for the modified bioelectrodes, i.e., electrocatalytic oxidation and reduction reaction, was carried out using linear sweep voltammetry, cyclic voltammetry, and open circuit potential. The overall performance of $\mu $ PAD-EBFC was evaluated using the polarization studies. Subsequently, the catalytic activity of enzymes on the electrode surface was validated by the scanning electron microscope. This simple and portable $\mu $ PAD-EBFC can generate the maximum power density to the order of $100~\mu \text{W}$ /cm2 ( $600~\mu \text{A}$ /cm2) at 0.505 V over prolonged durations of around 50 h. Hence, the presented $\mu $ PAD-EBFC shows good power density and stability, leading to its strong potential to power miniaturized microelectronics devices and sensors.

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.

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.

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.

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 NH2 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.

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.

Pitch deposits have negative effects on product quality, machine performance and production line profitability during pulp and paper manufacture. As traditional pitch control technology cannot provide satisfactory solutions in the pitch deposits, the enzymatic treatment has been rapidly developed for its high efficiency and pollution-free property. In this review, the chemical composition and present form of the pitch in pulp is first introduced, followed by a description of the pitch control enzymes. The emphasis is on the current research on enzymatic solutions to pitch problems, including the reaction mechanism, technology, and the present main problems of lipase, sterol esterases, laccase and lipoxygenase. Finally, the technology prospects in this field are proposed.在制浆造纸过程中，沉积的树脂会影响纸浆和成纸质量，降低设备的运行效率，最终造成经济损失。由于传统控制树脂障碍的方法不能很好地解决这一问题，因此具有高效催化和无污染的特性的生物酶法在该方面得到快速发展。文中介绍了树脂的成分、存在形式和控制树脂障碍的生物酶，重点介绍了脂肪酶、甾醇酯酶、漆酶和脂氧合酶控制纸浆中树脂障碍的机理和工艺研究方面的进展，指出了生物酶控制树脂障碍技术目前存在的主要问题，提出了该领域的主要研究方向，并对生物酶控制树脂障碍技术进行了展望。.

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.

Laccases are enzymes belonging to the group of oxidases. Laccases catalyze the oxidation of a variety of phenolic compounds, diamines and aromatic amines. Twenty-four fungal isolates were isolated from biodeteriorated ancient paper and parchment. These isolates were identified and found to belong to six genera: Alternaria, Aspergillus, Cladosporium, Penicillium, Rhizopus and Trichoderma, and were tested for producing laccase enzyme. Trichoderma harzianum have the ability of secreted laccase enzyme. The maximum production of laccase enzyme by T. harzianum was observed at 35 ○C and pH 5 after 6 days. The highest activity of laccase achieved at 35 ○C and pH 5 during the reaction. FTIR analysis revealed that the structure of extracted fungal pigments has aromatic ring and phenols group. Crude laccase was capable to decolorize different pigment structures. The enzyme showed great decolorization efficiency toward the extracted yellow pigment produced from Asp. terrus and Asp. ochareceous treated by 200 μl of partially purified enzyme. Laccase enzyme was used to decolorization pigment secreted from deteriorated pigmented fungi on paper and parchment during 30 days by using a pieces of paper and parchment inoculated by spore suspension. The results indicated that a high removal effect of fungal pigment on paper (71.21%) was recorded comparing to parchment samples (32.39%).

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.

High-resistance paper was manufactured by laccase-grafting of carboxymethyl cellulose (CMC) and chitosan (CPX) on Kraft pulp fiber. The reaction was mediated in the presence of laccase by one of the following polyphenols in the presence of air: gallic acid (GA), vanillic acid (VA) and catechol (1,2–DHB). Enzyme was added at constant loading (24 kg ton−1), 1% pulp consistency, 0.005% CMC, pH = 6.3 ± 0.5 and 2 mM of mediator. CPX content was assessed at two levels (0% and 0.005%). Treated pulps were analyzed by different mechanical tests (ring crush, mullen, corrugating medium test (CMT) flat crush of corrugating medium test and tension). An improvement in these parameters was obtained by biopolymer coupling and selected mediator. When using GA, three parameters increased more than 40%, while ring crush increased 120%. For the case of VA, properties were enhanced from 74% to 88% when CPX was added. For 1,2–DHB, there was not found a statistically significant difference between the results in the presence of CPX. Scanning electron microscopy, confocal microscopy, FTIR and 13C NMR were used in all papers in order to evaluate grafting. Hence, it was possible to correlate polymerization with an improvement of paper’s mechanical properties.

The competence of novel fungal consortium, consisting of Nigrospora sp. LDF00204 (accession no. KP732542) and Curvularia lunata LDF21 (accession no. KU664593), was investigated for the treatment of pulp and paper mill effluent. Fungal consortium exhibited enhanced biomass production under optimized medium conditions, i.e., glucose as carbon (C), sodium nitrate as nitrogen (N), C/N 1.5:0.5, pH 5, temperature 30 °C, and agitation 140 rpm, and significantly reduced biochemical oxygen demand (85.6%), chemical oxygen demand (80%), color (82.3%), and lignin concentration (76.1%) under catalytic enzyme activity; however, unutilized ligninolytic enzymes, such as laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP), were observed to be 13.5, 11.4, and 9.4 U/ml after the third cycle of effluent treatment in repeated batch process. Scanning electron microscopy (SEM) of fungal consortium revealed their compatibility through intermingled hyphae and spores, while the FTIR spectra confirmed the alteration of functional groups ensuring structural changes during the effluent treatment. Gas chromatography/mass spectroscopy (GC-MS) analysis showed the reduction of complex compounds and development of numerous low-molecular-weight metabolites, such as 1-3-dimethyl benzene, 2-chloro-3-methyl butane, pentadecanoic acid, and 1-2-benzene dicarboxylic acid, during the treatment, demonstrating the massive potential of the novel fungal consortium to degrade recalcitrant industrial pollutants.

This work addresses the development of a compact paper-based enzymatic microfluidic glucose/O2 fuel cell that can operate using a very limited sample volume (≈35 µl) and explores the energy generated by glucose at concentrations typically found in blood samples at physiological conditions (pH 7.4). Carbon paper electrodes combined with a paper sample absorption substrate all contained within a plastic microfluidic casing are used to construct the paper-based fuel cell. The anode catalysts consist of glucose dehydrogenase and [Os(4,4′-dimethoxy-2,2′-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator, while the cathode catalysts were bilirubin oxidase and [Os(2,2′-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator. The fuel cell delivered a linear power output response to glucose over the range of 2.5–30 mM, with power densities ranging from 20 to 90 µW cm−2. The quantification of the available electrical power as well as the energy density extracted from small synthetic samples allows planning potential uses of this energy to power different sensors and analysis devices in a wide variety of in-vitro applications., Peter Ó Conghaile is thanked for synthesis of the redox polymers. Neus Sabaté acknowledges funding from the European H2020 Framework Programme (Grant Agreement 648518 - SUPERCELL - ERC 2014 CoG). Juan Pablo Esquivel would like to thank the support from Marie Curie International Outgoing Fellowship (APPOCS) within the 7th European Community Framework Programme. F. Javier del Campo acknowledges funding from the Spanish Ministry of Economy through the DADDi2 project (TEC2013-48506-C3). Dónal Leech acknowledges support from Grant number 607793 under the Bioenergy FP7-PEOPLE-2013-ITN Programme.

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.

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.

An enzymatic biobleaching sequence (L VA QPO) 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.

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.