In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.     

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.     

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.     

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.     

Biological bleaching of chemical pulps

Use of biotechnology in pulp bleaching has attracted considerable attention and achieved interesting results in recent years. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes, xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. The aims of the enzymatic treatment depend on the actual mill conditions and may be related to environmental demands, reduction of chemical costs or maintenance or even improvement of product quality. The use of oxidative enzymes from white-rot fungi, that can directly attack lignin, is a second-generation approach, which could produce larger chemical savings than xylanase but has not yet been developed to the full scale. It is being studied in several laboratories in Canada, Japan, the U.S.A. and Europe. Certain white-rot fungi can delignify kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzyme manganese peroxidase and laccase can also delignify pulps and enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. Development work on laccase and manganese peroxidase continues. This article presents an overview of developments in the application of hemicellulase enzymes, lignin-oxidizing enzymes and white-rot fungi in bleaching of chemical pulps. The basic enzymology involved and the present knowledge of the mechanisms of the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.     

Effect of Organic Acids on Reactions Catalyzed by Manganese Peroxidase from Phanerochaete chrysosporium

The effect of several organic acids on the oxidation of Mn(II) catalyzed by manganese peroxidase was studied. Reactivities of manganese peroxidase and chemically prepared Mn(III) organic acid complexes towards phenolic compounds were compared. If lactate appears to be the best complexant for manganese peroxidase activity, chemically prepared Mn(III)—lactate complex is a less effective oxidant towards phenolic compounds than other Mn(III)—complexes. Our results agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. Malonate and lactate seem to be the most attractive complexants for practical applications of manganese peroxidase and were used in enzymatic treatment of hardwood kraft pulp. Bleaching of kraft pulp was studied and after alkaline extraction, a significant decrease of kappa number was measured. The bleaching was enhanced in lactate buffer.     

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and α-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached κ number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final κ number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by α-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA, gave the most effective κ number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability. Received: 16 August 1999 / Received revision: 13 December 1999 / Accepted: 15 December 1999     

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.     

Screening of white rot fungi for biobleaching of <Emphasis Type="Italic">Acacia</Emphasis> oxygen-delignified kraft pulp

To reduce the levels of chlorine-based chemicals in Acacia kraft pulp, we sought to isolate white rot fungus strains that could be used for biobleaching. For this purpose, we collected 600 fungal sources from Indonesia and subjected them to a three-step screening method. The first step involved culturing the strains on Acacia mangium wood powder, guaiacol and agar (WGA) medium. Of the 600 sources, 258 strains grew on WGA medium and generated a red color. The second step revealed that 31 of the 258 strains could degrade extractive-free A. mangium wood powder. The third step examined the ability of the strains to bleach A. mangium oxygen-delignified kraft pulp (A-OKP) under various pH conditions and showed that five strains could biobleach A-OKP at pH 5, 6, and 8. In contrast, the biobleaching abilities of Trametes versicolor and Phanerochaete chrysosporium, which served as standards, were much lower than those of the five new strains, particularly at pH 8. These five strains may be useful for biobleaching of A-OKP.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

Bioremediation of paper and pulp mill effluents

Pulp and paper mill effluents pollute water, air and soil, causing a major threat to the environment. Several methods have been attempted by various researchers throughout the world for the removal of colour from pulp and paper mill effluents. The biological colour removal process uses several classes of microorganisms--bacteria, algae and fungi--to degrade the polymeric lignin derived chromophoric material. White rot fungi such as Phanerochaete chrysosporium, Corius versicolor, Trametes versicolor etc., are efficient in decolourizing paper and pulp mill effluents. Gliocladium virens, a saprophytic soil fungus decolourised paper and pulp mill effluents by 42% due to the production of hemicellulase, lignin peroxidase, manganese peroxidase and laccase.     

Fiber surface characterization of old newsprint pulp deinked by combining hemicellulase with laccase-mediator system

Deinking of old newsprint (ONP) by combining hemicellulase with laccase-mediator system (LMS) was investigated, and surface chemical composition and fiber morphology changes during the deinking process were studied by electron spectroscopy for chemical analysis (ESCA), contact angle (CA), attenuated total reflectance fourier transform infrared spectrometry (ATR-FTIR), fiber quality analyzer (FQA), and environmental scanning electronic microscopy (ESEM). Results showed that, compared to the pulp deinked with hemicellulase or LMS individually, effective residual ink concentration (ERIC) was lower for the hemicellulase/LMS-deinked pulp. This indicated that there is a synergistic deinking effect between hemicellulase and LMS. It was found that O/C ratio of the fiber surface increased and the surface coverage of lignin decreased during the hemicellulase/LMS deinking process. The contact angle of the hemicellulase/LMS-deinked pulp was lower than that of pulps deinked with each individual enzyme. ESEM observations showed that more fibrils appeared on the fiber surface due to synergistic treatment.     

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     

Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö     

Xylanases in bleaching: From an idea to the industry

Abstract: The utilization of hemicellulases in bleaching of kraft (sulphate) pulp is considered as one of the most important new large-scale industrial applications of enzymes. This is partly due to the great potential of an environmentally safe method. This method has in a short period also proven to be economically realistic. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-/gb-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable. The hydrolysis of xylan or mannan in the inner fibre layers may also enhance the bleachability. In practical process conditions, properties of the enzymes such as substrate specificity and the pH and temperature optima are of utmost importance. The benefits obtained by enzymes are dependent on the chemical bleaching sequence used as well as on the residual lignin content of pulp. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process and consequently lowering the AOX of the effluents. Enzymes have been applied as a pretreatment both in conventional (C/D)EDED and in ECF (elementary chlorine-free) bleaching sequences. In the production of TCF (totally chlorine-free) pulps, enzymes have also been successfully used for increasing the brightness of pulp.     

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).     

Degradation of lignocellulose by extracellular enzymes produced by Thermomonospora fusca BD25

Degradation products from the addition of extracellular enzymes from Thermomonospora fusca BD25 to ball-milled wheat straw, oat spelt xylan and solubilised kraft pulps were characterised by HPLC and TLC. Overall, a high percentage hydrolysis of oat spelt xylan (28.9%) occurred after 26 h incubation. However, the rates of hydrolysis of ball-milled wheat straw and kraft pulp were approximately 4-6-fold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate was similar (22.2% and 25.9% respectively). Incubation of kraft pulp and ball-milled wheat straw by crude extracellular enzymes of T. fusca BD25 resulted in the detection of aromatic compounds at concentrations of 0.6 microg ml(-1) and 8.7 microg ml(-1), respectively. Hydrolysis of oat spelt xylan by T. fusca BD25 extracellular enzymes yielded a mixture of xylose, xylotriose and putative substituted-xylotriose, while the products of ball-milled wheat straw hydrolysis were xylose, glucose and a small oligomer present in the digest. The results highlight the ability of culture supernatant from T. fusca to release both simple sugars and aromatic compounds from lignocellulosic substrates and suggest a role for this organism in the biobleaching of pulp.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

Studies on decolourization, degradation and detoxification of chlorinated lignin compounds in kraft bleaching effluents by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora CZ-3, a wood degrading white rot fungus, was able to decolourize and degrade the first extraction stage effluent from kraft pulp bleaching at lower co-substrate concentration than the basidiomycetes previously investigated. With glucose at 1 g l⁻¹, this fungus removed up to 90% colour, 45% COD, 62% lignin, 32% AOX, and 36% EOX in 48 h at temperatures of 30–35°C and pH 4.0–4.5. In the absence of glucose, the fungus removed up to 62% of the colour. Significant reduction in chlorinated aromatic compounds was observed and toxicity to zebra fish was completely eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The molecular weight of chlorolignins was substantially reduced.     

Characterization of Palo Podrido, a Natural Process of Delignification in Wood

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of β-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the β-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.