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.     

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.     

Manganese Regulation of Veratryl Alcohol in White Rot Fungi and Its Indirect Effect on Lignin Peroxidase

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.     

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.     

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.     

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.     

New pulp biobleaching system involving manganese peroxidase immobilized in a silica support with controlled pore sizes

Attempts have been made to use manganese peroxidase (MnP) for chlorine-free pulp biobleaching, but they have not been commercially viable because of the enzyme's low stability. We developed a new pulp biobleaching method involving mesoporous material-immobilized manganese peroxidase from Phanerochaete chrysosporium. MnP immobilized in FSM-16, a folded-sheet mesoporous material whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability and tolerance to H(2)O(2). MnP immobilized in FSM-16 retained more than 80% of its initial activity even after 10 days of continuous reaction. We constructed a thermally discontinuous two-stage reactor system, in which the enzyme (39 degrees C) and pulp-bleaching (70 degrees C) reactions were performed separately. When the treatment of pulp with MnP by means of the two-stage reactor system and alkaline extraction was repeated seven times, the brightness of the pulp increased to about 88% within 7 h after completion of the last treatment.     

New Pulp Biobleaching System Involving Manganese Peroxidase Immobilized in a Silica Support with Controlled Pore Sizes

Attempts have been made to use manganese peroxidase (MnP) for chlorine-free pulp biobleaching, but they have not been commercially viable because of the enzyme's low stability. We developed a new pulp biobleaching method involving mesoporous material-immobilized manganese peroxidase from Phanerochaete chrysosporium. MnP immobilized in FSM-16, a folded-sheet mesoporous material whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability and tolerance to H₂O₂. MnP immobilized in FSM-16 retained more than 80% of its initial activity even after 10 days of continuous reaction. We constructed a thermally discontinuous two-stage reactor system, in which the enzyme (39°C) and pulp-bleaching (70°C) reactions were performed separately. When the treatment of pulp with MnP by means of the two-stage reactor system and alkaline extraction was repeated seven times, the brightness of the pulp increased to about 88% within 7 h after completion of the last treatment.     

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.     

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.     

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.     

Oxalic acid extraction as a posttreatment to increase the brightness of kraft pulps bleached by white-rot fungi

Pulp brightness gains obtained by fungal biobleaching are often decreased by dark MnO₂ deposits which are formed by the biologically-mediated oxidation of Mn(II). Oxalic acid extraction of O₂-delignified kraft pulps bleached by several white-rot fungi was found to increase pulp brightness by up to 9.7 ISO points due to MnO₂ dissolution. These results indicate that pulp extraction with oxalic acid can be used in order to accurately assess the net brightness gains achieved by fungal biobleaching.     

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 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.     

Synthesis of (1R,2S)-1-(3'-chloro-4' -methoxyphenyl)-1,2-propanediol (trametol) and (1R,2S)-1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol,chlorinated fungal metabolites in the natural environment

(1R,2S)-1-(3'-Chloro-4'-methoxyphenyl)-1,2propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.     

Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The recombinant laccase from Thermus thermophilus was applied to the biobleaching of wheat straw pulp. The best bleaching effect was when the pulp was treated with 3 U laccase g⁻¹ dry pulp at 90°C, pH 4.5, 8% consistency for 1.5 h. Under these conditions, the pulp brightness was increased by 3.3% ISO, and the pulp kappa number was decreased by 5.6 U. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp fibers. The use of enzyme-treated pulp saved 25% H₂O₂ consumption in subsequent peroxide bleaching without decreasing the final brightness. Pulp biobleaching in the presence of 5 mM ABTS further increased the pulp brightness by 1.5% ISO. This is the first report on the application of laccase from T. thermophilus in the pulp and paper sector.     

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.     

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.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.