Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi

Ethanol was produced by simultaneous saccharification and fermentation (SSF) from beech wood chips after bioorganosolve pretreatments by ethanolysis and white rot fungi, Ceriporiopsis subvermispora, Dichomitus squalens, Pleurotus ostreatus, and Coriolus versicolor. Beech wood chips were pretreated with the white rot fungi for 2-8 weeks without addition of any nutrients. The wood chips were then subjected to ethanolysis to separate them into pulp and soluble fractions (SFs). From the pulp fraction (PF), ethanol was produced by SSF using Saccharomyces cerevisiae AM12 and a commercial cellulase preparation, Meicelase, from Trichoderma viride. Among the four strains, C. subvermispora gave the highest yield on SSF. The yield of ethanol obtained after pretreatment with C. subvermispora for 8 weeks was 0.294 g g(-1) of ethanolysis pulp (74% of theoretical) and 0.176 g g(-1) of beech wood chips (62% of theoretical). The yield was 1.6 times higher than that obtained without the fungal treatments. The biological pretreatments saved 15% of the electricity needed for the ethanolysis.     

Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora

Methane fermentation of Japanese cedar wood was carried out after pretreatment with four strains of white rot fungi, Ceriporiopsis subvermispora ATCC 90467, CZ-3, CBS 347.63 and Pleurocybella porrigens K-2855. These fungi were cultivated on wood chip media with and without wheat bran for 4-8 weeks. The pretreated wood chip was fermented anaerobically with sludge from a sewage treatment plant. Pretreatments with C. subvermispora ATCC 90467, CZ-3 and CBS 347.63 in the presence of wheat bran for 8 weeks decreased 74-76% of beta-O-4 aryl ether linkages in the lignin to accelerate production of methane. After fungal treatments with C. subvermispora ATCC 90467 and subsequent 30-days methane fermentation, the methane yield reached 35 and 25% of the theoretical yield based on the holocellulose contents of the decayed and original wood, respectively. In contrast, treatment with the three strains of C. subvermispora without wheat bran cleaved 15-26% of the linkage and produced 6-9% of methane. There were no significant accelerating effects in wood chips treated with P. porrigens which has a lower ability to decompose the lignin. Thus, it was found that C. subvermispora, with a high ability to decompose aryl ether bonds of lignin, promoted methane fermentation of softwood in the presence of wheat bran.     

Biological degradation of tannins in sericea lespedeza (Lespedeza cuneata) by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus analyzed by solid-state 13C nuclear magnetic resonance spectroscopy.

Leaves of sericea lespedeza exhibit a high proportion of condensed tannin, resulting in poor forage quality. The white rot fungi Ceriporiopsis subvermispora and Cyathus sterocoreus are known to preferentially degrade lignin in a variety of plants and were evaluated for their ability to degrade condensed tannin from sericea leaves with the aim of improving digestibility. Relative levels of condensed tannin, cutin, pectin, and cellulose were monitored as a function of fungal treatment by solid-state cross-polarization and magic angle spinning 13C nuclear magnetic resonance spectroscopy. Total soluble phenolics, soluble tannins, and soluble and insoluble proanthocyanidin levels in fungus-treated and control samples were measured by established chemical techniques. Results indicate that both species of fungus preferentially degrade condensed tannin and that C. subvermispora is markedly superior to C. stercoreus in this capacity.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Biodegradation of lignocellulose in Bermuda grass by white rot fungi analyzed by solid-state 13C nuclear magnetic resonance.

Following the solid-state fermentation of Bermuda grass by two lignin-degrading white rot fungi, compositional changes have been observed in situ by utilization of cross-polarization and magic angle spinning 13C nuclear magnetic resonance difference spectra and interrupted decoupling spectra. Intensity differences in the 13C resonances assigned to specific components of the cell wall were used to observe these changes. Bermuda grass treated with Phanerochaete chrysosporium K-3 exhibited losses primarily in the polysaccharide components, with a smaller proportion of phenolic components also being degraded. In contrast, Ceriporiopsis subvermispora FP 90031-sp removed a proportionate amount of phenolic components compared with polysaccharide components. The results also indicated that C. subvermispora preferentially removes guaiacyl phenolic components relative to syringyl phenolic components, while P. chrysosporium was nonspecific in its attack on phenolic components.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Fungal resistance of rubber wood modified by fatty acid chlorides

Decay resistance of Rubber wood (Hevea brasiliensis) esterified with three fatty acid chlorides (hexanoyl chloride (C6), decanoyl chloride (C10) and tetra-decanoyl chloride (C14)) was evaluated. Unmodified and modified wood samples were exposed to a brown rot (Polyporus meliae) and a white rot (Coriolus versicolor) fungus for 12 weeks. Unmodified rubber wood was severely decayed by P. meliae and C. versicolor, which was indicated by significant weight loss. The rate of decay by brown rot was higher than white rot. Modified wood samples exhibited very good resistant to brown and white-rot fungi. The degree of protection increased with increase in degree of modification. P. meliae, a brown rot fungus, removed structural carbohydrate component in unmodified wood selectively whereas, C. vesicolor showed preference to lignin. The FTIR spectra of modified wood exposed to fungi show no significant changes in relative peak intensities of lignin/carbohydrates indicating effectiveness of chemically modified wood in restricting chemical degradation. Chemical modification occurred more efficiently at carbohydrate portion of the wood. Therefore, it is more effective in retarding decay due to P. meliae.     

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton NMR Spectra of 2,4-Dinitrophenyl Derivatives of Amino Acids

During the decay of wood by the typical white rot fungus Coriolus versicolor, Laccase III was the most abundantly secreted phenol oxidase. In this study, we proposed a possibility of the intermediate degradation steps of polymeric lignin by a purified Laccase III using synthetic [β-¹³C] and [β-¹⁴C]lignin (DHP). When the [β-¹⁴C]DHP was incubated with Laccase III, the water-soluble degradation product formed was about 8% of the applied [β-¹⁴C]DHP. The enzymic attack of Laccase III catalyzed the cleavage of the intermonomer linkages in the side chain structure of the polymeric lignin. In polymeric lignin metabolism by this fungus, laccase activity was closely related to the accumulation of water-soluble degradation products.     

Degradation of ferulic acid by a white rot fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Summary A ubiquitous white rot fungus Schizophyllum commune was used for the first time to study the degradation of ferulic acid. Vanillic acid was observed as one of the major products of ferulic acid catabolism, with vanillin formed as an intermediate. Almost 99.9% ferulic acid with a initial concentration of 5 mM was consumed by this fungus after 16 days of incubation at 37 °C.     

New polymeric model substrates for the study of microbial ligninolysis.

Lignin model dimers are valuable tools for the elucidation of microbial ligninolytic mechanisms, but their low molecular weight (MW) makes them susceptible to nonligninolytic intracellular metabolism. To address this problem, we prepared lignin models in which unlabeled and alpha-14C-labeled beta-O-4-linked dimers were covalently attached to 8,000-MW polyethylene glycol (PEG) or to 45,000-MW polystyrene (PS). The water-soluble PEG-linked model was mineralized extensively in liquid medium and in solid wood cultures by the white rot fungus Phanerochaete chrysosporium, whereas the water-insoluble PS-linked model was not. Gel permeation chromatography showed that P. chrysosporium degraded the PEG-linked model by cleaving its lignin dimer substructure rather than its PEG moiety. C alpha-C beta cleavage was the major fate of the PEG-linked model after incubation with P. chrysosporium in vivo and also after oxidation with P. chrysosporium lignin peroxidase in vitro. The brown rot fungus Gloeophyllum trabeum, which unlike P. chrysosporium lacks a vigorous extracellular ligninolytic system, was unable to degrade the PEG-linked model efficiently. These results show that PEG-linked lignin models are a marked improvement over the low-MW models that have been used in the past.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

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.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

白腐菌对染料脱色和降解作用的研究进展

白腐菌应用于废水处理始于二十世纪八十年代。本文对印染废水的处理方法、白腐菌及其对污染物的降解机理作了简要概述 ,着重介绍了白腐菌对染料脱色和降解作用的研究进展。白腐菌对染料的脱色解降作用机理有部分尚待进一步研究 ;同时 ,白腐菌的吸附作用亦不容忽视。     

Degradation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by the white rot fungus Phanerochaete sordida YK-624.

A method for the degradation of dioxins by white rot fungi was developed. Degradation of a mixture of 10 kinds of tetra- to octachlorodibenzo-p-dioxins (polychlorinated dibenzo-p-dioxins [PCDDs]) and tetra- to octachlorodibenzofurans (polychlorinated dibenzofurans [PCDFs]), which were chlorinated at 2-, 3-, 7-, and 8-positions of the molecules, by the white rot fungus Phanerochaete sordida YK-624 was studied in a stationary low-nitrogen medium. The percent degradation values of PCDDs and PCDFs were approximately 40 (tetra-chloro-) to 76% (hexachloro-) and 45 (tetrachloro-) to 70% (hexachloro-), respectively. Metabolites of 2,3,7,8-tetra- and octaCDD formed by P. sordida YK-624 included 4,5-dichlorocatechol and tetrachlorocatechol, respectively. These results suggest that white rot fungus is able to substantially degrade both PCDDs and PCDFs. This is the first report of the degradation of highly chlorinated PCDDs and PCDFs by a microorganism.     

Mineralization of Polycyclic Aromatic Hydrocarbons by the White Rot Fungus Pleurotus ostreatus

The white rot fungus Pleurotus ostreatus was able to mineralize to (sup14)CO(inf2) 7.0% of [(sup14)C]catechol, 3.0% of [(sup14)C]phenanthrene, 0.4% of [(sup14)C]pyrene, and 0.19% of [(sup14)C]benzo[a]pyrene by day 11 of incubation. It also mineralized [(sup14)C]anthracene (0.6%) much more slowly (35 days) and [(sup14)C]fluorene (0.19%) within 15 days. P. ostreatus did not mineralize fluoranthene. The activities of the enzymes considered to be part of the ligninolytic system, laccase and manganese-inhibited peroxidase, were observed during fungal growth in the presence of the various polycyclic aromatic hydrocarbons. Although activity of both enzymes was observed, no distinct correlation to polycyclic aromatic hydrocarbon degradation was found.