Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

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.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

Effects of Kraft Pulp and Lignin on Trametes versicolor Carbon Metabolism

The white rot basidiomycete Trametes (Coriolus) versicolor can substantially increase the brightness and decrease the lignin content of washed, unbleached hardwood kraft pulp (HWKP). Monokaryotic strain 52J was used to study how HWKP and the lignin in HWKP affect the carbon metabolism and secretions of T. versicolor. Earlier work indicated that a biobleaching culture supernatant contained all components necessary for HWKP biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital biobleaching system components renewed or replaced by the fungus. Nearly all of the CO₂ evolved by HWKP-containing cultures came from the added glucose, indicating that HWKP is not an important source of carbon or energy during biobleaching. Carbon dioxide appeared somewhat earlier in the absence of HWKP, but the culture partial O₂ pressure was little affected by the presence of pulp. The presence of HWKP in a culture markedly increased the culture's production of a number of acidic metabolites, including 2-phenyllactate, oxalate, adipate, glyoxylate, fumarate, mandelate, and glycolate. Although the total concentration of these pulp-induced metabolites was only 4.3 mM, these compounds functioned as effective manganese-complexing agents for the manganese peroxidase-mediated oxidation of phenol red, propelling the reaction at 2.4 times the rate of 50 mM sodium malonate, the standard chelator-buffer. The presence of HWKP in a culture also markedly stimulated fungal secretion of the enzymes manganese peroxidase, cellulase, and cellobiose-quinone oxidoreductase, but not laccase (phenol oxidase) or lignin peroxidase.     

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.     

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.     

Effects of Manganese Peroxidase on Residual Lignin of Softwood Kraft Pulp

Manganese peroxidase treatment lowered the kappa number of kraft pulp and increased the alkali extractability of the residual lignin but did not directly solubilize it. This indicates that MnP partially oxidizes the lignin in the pulp but does not degrade it to soluble fragments.     

Relationship between Fungal Biomass Production and the Brightening of Hardwood Kraft Pulp by Coriolus versicolor

The white-rot fungus Coriolus versicolor increased the brightness of hardwood kraft pulp by two mechanisms depending on the concentration of available nitrogen. In low-nitrogen conditions, the brightening process was a chemical effect mediated by the fungus, associated with the removal of residual lignin in the pulp; kappa number was used as an indicator of lignin concentration. A five-day treatment in low-nitrogen conditions increased the brightness of hardwood kraft pulp from 36.2 to 54.5%, with a corresponding decrease in kappa number from 12.0 to 8.5, equivalent to a reduction in the lignin concentration from ca. 2.0% (wt/wt) to ca. 1.4% (wt/wt). Under these conditions, we concluded that the brightening of the pulp was a secondary metabolic event initiated after the depletion of available nitrogen. This method of brightening has been described as bleaching or biobleaching. By contrast, in high-nitrogen conditions, the brightening was a physical effect associated with the dilution of the dark pulp fibers by the relatively high levels of brighter fungal mycelium produced. Since this method of brightening was not evidently associated with lignin removal, it cannot be described as bleaching. In pulp samples brightened in high-nitrogen conditions, as brightness increased, there was a corresponding increase in kappa number. This observation was explained by the consumption of potassium permanganate by the fungal mycelium, which interfered with kappa number determinations at high fungal biomass levels.     

Reactivities of Various Mediators and Laccases with Kraft Pulp and Lignin Model Compounds

Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification.     

Improving the bioremediation of phenolic wastewaters by Trametes versicolor

The successful bioremediation of a phenolic wastewater by Trametes versicolor was found to be dependent on a range of factors including: fungal growth, culture age and activity and enzyme (laccase) production. These aspects were enhanced by the optimisation of the growth medium used and time of addition of the pollutant to the fungal cultures. Different media containing 'high' (20 g/L), 'low' (2 g/L) and 'sufficient' (10 g/L) concentrations of carbon and nitrogen sources were investigated. The medium containing both glucose and peptone at 10 g/L resulted in the highest Growth Related Productivity (the product of specific yield and micro) of laccase (1.46 Units of laccase activity)/gram biomass/day and was used in all further experiments. The use of the guaiacol as an inducer further increased laccase activity 780% without inhibiting growth; similarly the phenolic effluent studied boosted activity almost 5 times. The timing of the addition of the phenolic effluent was found to have important consequences in its removal and at least 8 days of prior growth was required. Under these conditions, 0.125 g phenol/g biomass and 0.231 g o-cresol/g biomass were removed from solution per day.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

Transformation of triclosan by Trametes versicolor and Pycnoporus cinnabarinus

We investigated the ability of Trametes versicolor and Pycnoporous cinnabarinus to metabolize triclosan. T. versicolor produced three metabolites, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-xylopyranoside, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-glucopyranoside, and 2,4-dichlorophenol. P. cinnabarinus converted triclosan to 2,4, 4'-trichloro-2'-methoxydiphenyl ether and the glucoside conjugate known from T. versicolor. The conjugates showed a distinctly lower cytotoxic and microbicidal activity than triclosan did.     

变色栓菌产锰过氧化物酶的条件优化

研究了多种培养基组分及培养条件对变色栓菌产锰过氧化物酶(MnP)的影响.当培养基中果糖浓度为20g/L,酒石酸铵浓度为10mmoL/L,吐温80浓度为1.0g/L,MgSO4·7H2O为0.43g/L,最终pH为4.5,500mL三角瓶装液量为100mL,接种量为10片(φ8mm)菌苔,培养温度为30℃,转速为280r/min时,MnP的活力有了很大程度的提高,最高酶活力可达2,270U/L.     

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.     

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 degrees C for 3 days, and then at 30 degrees C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor

Lignite (brown coal) can be liquefied/solubilized with several fungi by different mechanisms. When applied industrially, only catalytic mechanisms can compete with chemical methods. The well-known fungal ligninolytic peroxidases are at a disadvantage, in that the relatively expensive hydrogen peroxide must be used as a cofactor. Comparing several fungal strains, we observed that the fungus Trametes versicolor is able to decolorize coal-derived humic acids, producing a considerable amount of laccase in the process. During this reaction the amount of humic acids decreases whilst that of fulvic acids increases; this was verified by optical density measurement and GPC after the two substance classes had been separated. Received: 27 August 1998 / Received revision: 4 November 1998 / Accepted: 7 November 1998     

云芝菌株遗传多样性的ISSR分析

来自全国的34株野生菌株经形态学特征和结合ITS序列鉴定为云芝。采用ISSR标记技术对34株野生云芝菌株进行遗传多样性分析。从20条引物中筛选出7条ISSR引物,扩增得到95个扩增位点,其中多态性位点88个。多态性位点占92.6%,表明ISSR标记的多态性非常高。基于ISSR条带构建亲缘关系树状图,其中遗传变异系数范围为0.58-0.91。34个云芝菌株在相似系数0.60时分为4个类群,不同菌株的遗传差异性与地理分布有一定联系。     

Isolation and Identification of the Coal-Solubilizing Agent Produced by Trametes versicolor

Low-ranked coals were dissolved by using cell extracts derived from liquid cultures of Trametes versicolor. The coal-solubilizing agent (CSA) was separated from the broth components by a multistep isolation procedure including reverse-phase high-pressure liquid chromatography, size exclusion chromatography, ethanol fractionation, and recrystallization. Staircase voltammetry was used to show that two CSA moieties can coordinate to aqueous copper(II) ion. A molecular weight determination (using amperometry) gave an apparent molecular weight of 1.3₄ × 10² g/mol ± 8%. Nuclear magnetic resonance indicated that all protons on CSA are exchangeable in D₂O and that there is only one type of carbon in CSA. The infrared spectrum of recrystallized CSA is identical to that of ammonium oxalate, and X-ray studies confirmed the crystal structure and composition of CSA to be that of ammonium oxalate monohydrate. The equivalent weight of the coal in solution, when the coal was dissolved by ammonium oxalate, is 7,940 g of coal per mol of iron present in the coal.