Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase Secreted from Phanerochaete sordida YK-624

In vitro bleaching of an unbleached hardwood kraft pulp was performed with manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624. When the kraft pulp was treated with partially purified MnP in the presence of MnSO₄, Tween 80, and sodium malonate with continuous addition of H₂O₂ at 37°C for 24 h, the pulp brightness increased by about 10 points and the kappa number decreased by about 6 points compared with untreated pulp. The pulp brightness was also increased by 43 points to 75.5% by multiple (six) treatments with MnP combined with alkaline extraction. Our results indicate that in vitro degradation of residual lignin in hardwood kraft pulp with MnP is possible.     

Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp

Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening.     

Investigation of hydroxamic acids as laccase-mediators for pulp bleaching

A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied.     

Immobilization of a Thermostable Cellobiose 2-Epimerase from Rhodothermus marinus JCM9785 and Continuous Production of Epilactose

Previous study has shown that a crude manganese peroxidase (MnP) preparation from the fungus could bleach oxygen-alkaline treated hardwood kraft pulp (OKP) with manganese, glucose, and glucose oxidase. Using purified MnP instead of the crude one also did OKP bleaching with Tween 20. We conclude that MnP is important in this fungal bleaching system.     

Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase from Phanerochaete sordida YK-624 without Addition of MnSO(inf4)

In vitro bleaching of an unbleached hardwood kraft pulp was performed with partially purified manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624 without the addition of MnSO(inf4) in the presence of oxalate, malonate, or gluconate as manganese chelator. When the pulp was treated without the addition of MnSO(inf4), the pulp brightness increased by about 10 points in the presence of 2 mM oxalate, but the brightness did not significantly increase in the presence of 50 mM malonate, a good manganese chelator. Residual MnP activity decreased faster during the bleaching with MnP without MnSO(inf4) in the presence of malonate than in the presence of oxalate. Oxalate reduced MnO(inf2) which already existed in the pulp or was produced from Mn(sup2+) by oxidation with MnP and thus supplied Mn(sup2+) to the MnP system. The presence of gluconate, produced by the H(inf2)O(inf2)-generating enzyme glucose oxidase, also improved the pulp brightness without the addition of MnSO(inf4), although treatment with gluconate was inferior to that with oxalate with regard to increase of brightness. It can be concluded that bleaching of hardwood kraft pulp with MnP, using manganese originally existing in the pulp, is possible in the presence of oxalate, a good manganese chelator and reducing reagent.     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Production and Characterization of Trametes versicolor Mutants Unable To Bleach Hardwood Kraft Pulp

Protoplasts of the monokaryotic strain 52J of Trametes versicolor were treated with UV light and screened for the inability to produce a colored precipitate on guaiacol-containing agar plates. Mutants unable to oxidize guaiacol had absent or very low secretion of laccase and manganese peroxidase (MnP) proteins. All isolates unable to secrete MnP were also unable to bleach or delignify kraft pulp. One mutant strain, M49, which grew normally but did not oxidize guaiacol, was tested further with a number of other substrates whose degradation has been associated with delignification by white rot fungi. Compared with the parent, 52J, mutant M49, secreting no MnP and low laccase, could not brighten or delignify kraft pulp, produced less ethylene from 2-keto methiolbutyric acid, released much less (sup14)CO(inf2) from [(sup14)C]DHP (a synthetic lignin-like polymerizate), and produced much less methanol from pulp. This mutant also displayed decreased abilities to oxidize the dyes poly B-411, poly R-478, and phenol red compared with the wild-type strain and was also unable to decolorize kraft bleachery effluent or mineralize its organochlorine. Addition of purified MnP in conjunction with H(inf2)O(inf2), MnSO(inf4), and an Mn(III) chelator to M49 cultures partially restored methanol production, pulp delignification, and biobleaching in some cases.     

Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.

Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.     

Studies on mediators of manganese peroxidase for bleaching of wood pulps

In order to enhance the bleaching effect of manganese peroxidase (MnP), unsaturated fatty acids, thiol-containing compounds and various other organic compounds were applied in pulp bleaching experiments with MnP. Thiol-containing compounds did not improve the pulp bleaching effect by MnP. Some unsaturated fatty acids, linoleic acid and linolenic acid provided a better pulp bleaching effect than Tween 80. The correlation between the number of C=C bonds in a fatty acid and its pulp bleaching effect was also investigated. The MnP pulp bleaching capability was shown to depend on the carboxylic acid used. A combination of Tween 80 and a carboxylic acid resulted in higher pulp brightness than that obtained with Tween 80 alone. A laccase mediator, 3-hydroxy-1,2,3-benzotriazin-4(3H)-one, could also enhance the MnP pulp bleaching effect.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase–HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase–HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase–HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols–filipin signals were almost completely absent.     

Influence of the cycle number in processing of cellulose from waste paper on its ability to hydrolysis by cellulases

Hydrolytic ability of laboratory enzyme preparations from fungus of the Penicillium genus was investigated using kraft pulp from nonbleached softwood and bleached hardwood cellulose as substrates. The enzyme preparations were shown to efficiently hydrolyze both softwood and hardwood cellulose. The yields of glucose and reducing sugars were 24–36 g/l and 27–37 g/l from 100 g/l of dry substrate in 48 h, respectively, and depended on the number of substrate grinding cycles.     

Changes in isozyme patterns between monokaryons and dikaryons of a bipolar Coprinus

Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum. It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp.     

Prebleaching of kraft pulp with full-length and truncated forms of a thermostable modular xylanase from Rhodothermus marinus

Full-length and truncated forms of a modular thermostable xylanase (EC 3.2.1.8., glycoside hydrolase family 10) were used in bleaching sequences of hardwood and softwood kraft pulps. Enzymatic treatment led to brightness gains of all pulps but the result depended on the pulp source. The presence of the additional domains in the full-length enzyme (including carbohydrate-binding modules) did not improve the bleaching process. No significant change in viscosity was seen after enzyme treatments indicating an unaffected pulp fibre length.     

A Possible Role of Manganese Peroxidase during Biobleaching by the Pulp Bleaching Fungus SKB-1152

The fungus SKB-1152 bleaches oxygen-alkaline treated hard wood kraft pulp (OKP) rapidly. In the initial phase of fungal treatment, maximum production of manganese peroxidase (MnP) was observed. The filtrate from a 1-day fungal treatment could bleach OKP when manganese, glucose, and glucose oxidase were added. A possible role of MnP in the initial fungal bleaching process is suggested.     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Fate of Residual Lignin during Delignification of Kraft Pulp by Trametes versicolor

The fungus Trametes versicolor can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing ¹⁴C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with T. versicolor. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO₂ varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the T. versicolor cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase-HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase-HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase-HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols-filipin signals were almost completely absent.     

Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)

Summary Bleaching of hardwood kraft pulp by Trametes versicolor was accompanied by release and accumulation of methanol, which was produced by demethylation of the pulp. A partial demethylation of the pulp was observed with isolated laccase I from T. versicolor. The extent of demethylation by laccase was increased to the level released by the fungus by addition of 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Methanol release by the laccase/ABTS combination was followed by slower kappa reduction. Both methanol release and kappa reduction were dependent on laccase and ABTS concentrations. The fungus did not produce a stable equivalent of ABTS during bleaching, because extracellular culture fluid from bleaching cultures gave only the same methanol release from pulp as laccase I. Pulp viscosity, an indicator of cellulose chain length, was decreased only slightly by laccase. Thus the enzyme in the presence of ABTS, unlike the fungus, specifically attacks lignin.Offprint requests to: R. Bourbonnais     

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