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.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

Biodecolorization screening of synthetic dyes by four white-rot fungi in a solid medium: possible role of siderophores

AIMS: Four selected fungi were screened for their ability to decolourize a textile effluent and commercial reactive dyes in a solid medium. METHODS AND RESULTS: Ligninolytic enzymes activities (lignin peroxidase, manganese peroxidase and laccase) and siderophores presence were monitored in decolourized plates. RESULTS: The results showed low lignin peroxidase activity and no manganese peroxidase activity was detected for all fungi. Laccase activity was observed in Reactive Blue 19 decolourized plates by Trametes versicolor and Trametes villosa. Siderophores presence was observed in Trametes versicolor, Phanerochaete chrysosporium and Lentinus edodes decolourized plates. CONCLUSION: Lentinus edodes displayed the greatest decolourization ability both in terms of extent and rapidity of decolourization. SIGNIFICANCE AND IMPACT OF THE STUDY: The transformation observed for dyes open the possibility to study siderophores to treat dyes and textile effluents.     

Enzymatic activity, osmotic stress and degradation of pesticide mixtures in soil extract liquid broth inoculated with Phanerochaete chrysosporium and Trametes versicolor

In this study we examined the extracellular enzymatic activity of two white rot fungi (Phanerochaete chrysosporium and Trametes versicolor) in a soil extract broth in relation to differential degradation of a mixture of different concentrations (0-30 p.p.m.) of simazine, dieldrin and trifluralin under different osmotic stress (-0.7 and -2.8 MPa) and quantified enzyme production, relevant to P and N release (phosphomonoesterase, protease), carbon cycling (beta-glucosidase, cellulase) and laccase activity, involved in lignin degradation. Our results suggest that T. versicolor and P. chrysosporium have the ability to degrade different groups of pesticides, supported by the capacity for expression of a range of extracellular enzymes at both -0.7 and -2.8 MPa water potential. Phanerochaete chrysosporium was able to degrade this mixture of pesticides independently of laccase activity. In soil extract, T. versicolor was able to produce the same range of enzymes as P. chrysoporium plus laccase, even in the presence of 30 p.p.m. of the pesticide mixture. Complete degradation of dieldrin and trifluralin was observed, while about 80% of the simazine was degraded regardless of osmotic stress treatment in a nutritionally poor soil extract broth. The capacity of tolerance and degradation of high concentrations of mixtures of pesticides and production of a range of enzymes, even under osmotic stress, suggest potential bioremediation applications.     

Degradation of anthracene by selected white rot fungi

Abstract Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor , AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona , AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium .     

Biodegradation of soil humic acids by Streptomyces viridosporus.

Biodegradation of soil humic acids by Streptomyces viridosporus ATCC 39115 growing in a mineral salts--glucose medium was demonstrated. This biodegradation accompanies bacterial growth and is, therefore, presumed to be a primary metabolic activity, but humic acids were not used as the sole source of carbon. This bacterial activity was enhanced when cells were shaken and within a pH range of 6.5-8.5. In further experiments, the relative abilities of S. viridosporus to mineralized [14C]melanoidin, used as synthetic humic acid, were also established. In contrast to the white rot fungus Phanerochaete chrysosporium, another microorganism exhibiting humic acid degrading activity at acidic pH, poor extracellular activities were found in culture medium of S. viridosporus, and veratryl alcohol does not result in increased humic acid degradation. In spite of some peroxidase activity measured in culture filtrates and analyzed by polyacrylamide gel electrophoresis, the humic acid degrading system of S. viridosporus, in these experimental conditions, seems to be cell associated.     

Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes

Two closely linked lignin peroxidase (LPO)-encoding genes (lpo) from Phanerochaete chrysosporium were isolated. Nucleotide sequence studies indicated that the two genes are separated by 1.3 kb of flanking DNA and transcribed in opposite directions. Cloned P. chrysosporium lpo gene probes have been shown to hybridize to multiple sequences present in the DNAs of the white-rot fungi, Bjerkandera adusta, Coriolus versicolor and Fomes lignosus, but no hybridization was detected with DNA from Pleurotus ostreatus. Thus, lpo gene families appear to be common in a number of lignin-degrading basidiomycetes, some of which have not yet been shown to produce LPO proteins.     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Trametes versicolor ligninase: isozyme sequence homology and substrate specificity

The substrate specificity of three ligninase isozymes from the white-rot fungus Trametes versicolor has been investigated using stereochemically defined synthetic dimeric models for lignin. The isozymes have been found to attack non-phenolic beta-O-4 as well as beta-1 lignin model compounds. This finding confirms the classification of the isozymes from T. versicolor as ligninases. The amino-terminal residues of the three isozymes from T. versicolor have been determined using Edman degradation. Minor differences found between the sequences suggest the existence of several structural genes for ligninase in T versicolor. Comparisons have been made with the sequences of three previously reported ligninases from Phanerocompaete chrysosporium, another lignin-degrading fungus. One of the sequences from P. chrysosporium is distinctly more similar to the T. versicolor isozymes than to the other two sequences from P. chrysosporium.     

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.     

Estrogenic reduction of styrene monomer degraded by Phanerochaete chrysosporium KFRI 20742

The characteristic biodegradation of monomeric styrene by Phanerochaete chrysosporium KFRI 20742, Trametes versicolor KFRI 20251 and Daldinia concentrica KFRI 40-1 was carried out to examine the resistance, its degradation efficiency and metabolites analysis. The estrogenic reduction effect of styrene by the fungi was also evaluated. The mycelium growth of fungi differentiated depending on the concentration levels of styrene. Additionally P. chrysosporium KFRI 20742 showed superior mycelium growth at less than 200 mg/l, while D. concentrica KFRI 40-1 was more than 200 mg/l. The degradation efficiency reached 99% during one day of incubation for all the fungi. Both manganese-dependent peroxidase and laccase activities in liquid medium were the highest at the initial stage of incubation, whereas the lowest was after the addition of styrene. However, both activities were gradually recovered after. The major metabolites of styrene by P. chrysosporium KFRI 20742 were 2-phenyl ethanol, benzoic acid, cyclohexadiene-1,4-dione, butanol and succinic acid. From one to seven days of incubating the fungi, the expression of pS2 mRNA widely known as an estrogen response gene was decreased down to the level of baseline after one day. Also, the estrogenic effect of styrene completely disappeared after treatment with supernatant of P. chrysosporium KFRI 20742 from one week of culture down to the levels of vehicle.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

Solubilization and Mineralization of Lignin by White Rot Fungi

The white rot fungi Lentinula edodes, Phanerochaete chrysosporium, Pleurotus sajor-caju, Flammulina velutipes, and Schizophyllum commune were grown in liquid media containing ¹⁴C-lignin-labelled wood, and the formation of water-soluble ¹⁴C-labelled products and ¹⁴CO₂, the growth of the fungi, and the activities of extracellular lignin peroxidase, manganese peroxidase, and laccase were measured. Conditions that affect the rate of lignin degradation were imposed, and both long-term (0- to 16-day) and short-term (0- to 72-h) effects on the production of the two types of product and on the activities of the enzymes were monitored. The production of ¹⁴CO₂-labelled products from the aqueous ones was also investigated. The short-term studies showed that the different conditions had different effects on the production of the two products and on the activities of the enzymes. Nitrogen sources inhibited the production of both products by all species when differences in growth could be discounted. Medium pH and manganese affected lignin degradation by the different species differently. With P. chrysosporium, the results were consistent, with lignin peroxidase playing a role in lignin solubilization and manganese peroxidase being important in subsequent CO₂ production.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.     

Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium

The biological transformation of lignocellulose of Achras zapota by white rot fungi, Phanerochaete chrysosporium, in solid state fermentation (SSF) was studied for 28 days. The kinetic transformation of lignocellulose was monitored through the determination of acid soluble and acid insoluble lignin content, total organic carbon (TOC) and chemical oxygen demand (COD). The lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) were quantified on weekly intervals. The degradation of lignin and other structural moieties of A. zapota lignocellulose were confirmed by high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The degradation of lignin was increased after 7 days of fermentation with the release of water soluble and fermentable products. The LiP and MnP activities were increased in the first week of SSF and lignin degradation was also set to increase. This was accompanied with increase in COD by 94.6% and TOC by 80% and lignin content was decreased by 76%. The maximum activities of the enzymes LiP and MnP in extracellular fluid of SSF under nitrogen limitation, at pH 5.0, at temperature 37 degrees C and at 60% humidity were 2100 U/L and 1200 U/L.     

Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767

It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2(prm1)-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M(infr) of 46,500.