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.     

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.     

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.     

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     

Mineralization of 14C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

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.     

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.     

Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi

Two families of peroxidases—lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)—are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of ¹⁴CO₂ from ¹⁴C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of ¹⁴CO₂ release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.     

Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora

The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize ¹⁴C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly.     

Role of Organic Acids in the Manganese-Independent Biobleaching System of Bjerkandera sp. Strain BOS55

Bjerkandera sp. strain BOS55 is a white rot fungus that can bleach EDTA-extracted eucalyptus oxygen-delignified kraft pulp (OKP) without any requirement for manganese. Under manganese-free conditions, additions of simple physiological organic acids (e.g., glycolate, glyoxylate, oxalate, and others) at 1 to 5 mM stimulated brightness gains and pulp delignification two- to threefold compared to results for control cultures not receiving acids. The role of the organic acids in improving the manganese-independent biobleaching was shown not to be due to pH-buffering effects. Instead, the stimulation was attributed to enhanced production of manganese peroxidase (MnP) and lignin peroxidase (LiP) as well as increased physiological concentrations of veratryl alcohol and oxalate. These factors contributed to greatly improved production of superoxide anion radicals, which may have accounted for the more extensive biobleaching. Optimum biobleaching corresponded most to the production of MnP. These results suggest that MnP from Bjerkandera is purposefully produced in the absence of manganese and can possibly function independently of manganese in OKP delignification. LiP probably also contributed to OKP delignification when it was present.     

Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi

Manganese dependent peroxidase (MnP) is the main enzyme implicated in the biobleaching of kraft pulps by white rot fungi. The goal of this study was to evaluate the Mn requirement for biobleaching of eucalyptus oxygen delignified kraft pulp (OKP) by various white rot fungi: Trametes versicolor, Phanerochaete sordida, Phlebia radiata, Stereum hirsutum and Bjerkandera sp. strain BOS55. All of the strains tested produced MnP and provided extensive bleaching of OKP when 33 μM Mn was included in the medium. Bjerkandera sp. strain BOS55 was the only strain that also displayed MnP production and biobleaching activity of EDTA-extracted OKP in the complete absence of Mn. However, MnP and biobleaching activity in the absence of Mn was dependent on the presence of organic acids in the medium. The fact the biobleaching was correlated to MnP activity irrespective of whether Mn was present or absent suggests that there may be roles for MnP in Bjerkandera under Mn-deficient conditions. Although manganese-independent peroxidase (MIP) and lignin peroxidase (LiP) were also detected, the titres were much smaller in comparison with those of MnP, so their relative role in biobleaching can be predicted to have a minor importance in comparison with MnP. Only in the case of Bjerkandera, was the expression of LiP stimulated in the presence of oxalate but final brightness was not substantially affected.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

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.     

Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata

 The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of ¹⁴C-ring-labelled synthetic lignin ([¹⁴C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures when malonate was omitted. Degradation of [¹⁴C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of MnP and LiP expression and have different roles in the degradation of lignin by P. radiata. Received: 30 August 1995/Received revision: 10 January 1996/Accepted: 12 February 1996     

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.     

The effect of manganese on the production of Phanerochaete flavido-alba ligninolytic peroxidases in nitrogen limited cultures

Manganese supplementation of culture medium affected Phanerochaete flavido-alba FPL 106507 growth, glucose consumption and extracellular protein accumulation. Both the titre and time of detection of lignin peroxidase (LiP) were affected by manganese concentration in the medium, whereas with manganese peroxidase (MnP) only the titre was affected. In high Mn(II) containing cultures highest manganese peroxidase levels and a decrease in extracellular veratryl alcohol accumulation were observed. After FPLC a number of haemprotein peaks showing manganese peroxidase activity were detected in Mn(II) supplemented cultures. On the contrary, only haemprotein peaks of lignin peroxidase were detected in culture medium not supplemented with Mn(II).     

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.     

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.     

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.     

Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55

Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1⁻¹) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1⁻¹. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.