Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degrees C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus.     

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.     

Decolorization of simulated textile dye baths by crude laccases from Trametes hirsuta and Cerrena unicolor

In this study crude laccases from the white‐rot fungi Cerrena unicolor and Trametes hirsuta were tested for their ability to decolorize simulated textile dye baths. The dyes used were Remazol Brilliant Blue R (RBBR) (100 mg/L), Congo Red (12.5 mg/L), Lanaset Grey (75 mg/L) and Poly R‐478 (50 mg/L). The effect of redox mediators on dye decolorization by laccases was also assessed. C. unicolor laccase was able to decolorize all the dyes tested. It was especially effective towards Congo Red and RBBR with 91 and 80% of color removal in 19.5 h despite the fact that simulated textile dye baths were used. Also Poly R‐478 and Lanaset Grey were partially decolorized (69 and 48%, respectively). C. unicolor laccase did not need any mediators for removing the dyes. However, T. hirsuta laccase was only able to decolorize simulated Congo Red and RBBR dye baths (91 and 45%, respectively) in 19.5 h without mediators. When using mediators the decolorization capability was enhanced substantially, e.g. Poly R‐478 was decolorized by 78% in 25.5 h. On the whole, both laccases showed potential to be used in industrial applications.     

Decolorization of Several Polymeric Dyes by the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

The polymeric dyes Poly B-411, Poly R-481, and Poly Y-606 were examined as possible alternatives to the radiolabeled lignin previously used as a substrate in lignin biodegradation assays. Like lignin degradation, the decolorization of these dyes by the white rot basidiomycete Phanerochaete chrysosporium occurred during secondary metabolism, was suppressed in cultures grown in the presence of high levels of nitrogen, and was strongly dependent on the oxygen concentration in the cultures. A variety of inhibitors of lignin degradation, including thiourea, azide, and 4′-O-methylisoeugenol, also inhibited dye decolorization. A pleiotropic mutant of P. chrysosporium, 104-2, lacking phenol oxidase and ligninolytic activity was also not able to decolorize the polymeric dyes, whereas a phenotypic revertant strain, 424-2, regained this capacity. All of these results suggest that the ligninolytic degradation activity of the fungus was responsible for the decolorization of these dyes.     

Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

Summary The potential of a recently isolated wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the fungus proved to be efficiently capable of both biodegradation and biosorption as the major dye removal mechanisms. The extent of biodegradation was associated with the levels of the extracellular ligninolytic enzymes such as manganese peroxidase and laccase.     

Decolorization of synthetic dyes by<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> isolates differing in ligninolytic properties

The ability to decolorize four synthetic dyes (Phenol Red, Evans Blue, Eosin Yellowish and Poly B411) in fivePleurotus ostreatus strains (a parental strain and four isolates derived from it) was determined. Two of the isolates had markedly higher and other two substantially lower production of ligninolytic enzymes and hydrogen peroxide that the parental strain. Like the parental strain, the higher-producing isolates were able to decolorize all the tested dyes, but not to a higher extent than the parental strain. In contrast, two lower-producing isolates exhibited slow decolorization, which was incomplete even at the end of cultivation. Evans Blue and Eosin Yellowish strongly suppressed the growth of the strains, while Phenol Red and Poly B411 induced none or only a very slight growth reduction.     

Decolorization of Remazol Brilliant Blue (RBBR) and Poly R-478 dyes by <Emphasis Type="Italic">Bjerkandera adusta</Emphasis> CCBAS 930

An anamorphic Bjerkandera adusta CCBAS 930 strain isolated from soil was found to decolorize two anthraquinonic dyes: Remazol Brilliant Blue R and Poly R-478. The reduction in the level of phenolic compounds in liquid B. adusta cultures containing RBBR and Poly R-478 was correlated with decolorization of studied dyes, which suggested their biodegradation. It was shown that this process was coupled with induction of secondary metabolism (idiophase) and peak peroxidase activity in culture medium, and the appearance of aerial mycelium. Decolorization of dyes depended on the presence of glucose (cometabolism).     

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H₂O₂ resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.     

In vivo decolourization of the polymeric dye poly R‐478 by corncob cultures of Phanerochaete chrysosporium

In this paper, the in vivo decolourization of the polymeric dye Poly R‐478 by semi‐solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l). Maximum manganese‐dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures. The polymeric dye Poly R‐478 (0.02 w/v) was added to three‐day‐old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO₂ cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R‐478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation. In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above‐mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R‐478.     

Decolorization of polymeric dyes by a novel Penicillium isolate

A novel polymeric dye-degrading fungal strain ATCC 74414 was isolated. Taxonomic identification including morphological and cultural characterization indicated that this isolate was a strain of Penicillium. Strain ATCC 74414 aerobically decolorized both Poly R-478 and Poly S-119 in liquid media containing 0.01% of polymeric dyes. The decolorization rate was examined in three distinct liquid media: Schenk and Hildebrandt-K₂SO₄ medium (SHK), potato dextrose broth (PDB), and half Murashige-Skoog medium (HMS). Strain ATCC 74414 rapidly decolorized R-478 in SHK medium but the color was subsequently released from the mycelial mass into the medium after 2–3 days, indicating that the decolorization in SHK medium could be due to adsorption of Poly R-478 by the mycelia. In contrast, in HMS and PDB media ATCC 74414 decolorized Poly R-478 more steadily, and the dye was initially adsorbed onto the mycelia and was subsequently decolorized without being released into the medium. Strain ATCC 74414 also decolorized Poly S-119 steadily in SHK, HMS and PDB media. It appears that the decolorization process involved initial mycelial adsorption of dye compounds, which was probably followed by biodegradation through microbial metabolism, and the decolorization may be affected by medium constituents. Although aerobic decolorization may not necessarily lead to complete mineralization of dyes, these results have suggested the potential of strain ATCC 74414 in bioremediation of dye-contaminated water and soil.     

White-rot fungi capable of decolourising textile dyes under alkaline conditions

Twelve white-rot fungal strains belonging to seven different species were screened on plates under alkaline condition to study the decolourisation of the textile dyes Reactive Black 5 and Poly R-478. Three strains of Trametes versicolor (Micoteca da Universidade do Minho (MUM) 94.04, 04.100 and 04.101) and one strain of Phanerochaete chrysosporium (MUM 94.15) showed better decolourisation results. These four strains were used for decolourisation studies in liquid culture medium. All four selected strains presented more efficient decolourisation rates on Reactive Black 5 than on Poly R-478. For both dyes on solid and liquid culture media, the decolourisation capability exhibited by these strains depended on dye concentration and pH values of the media. Finally, the decolourisation of Reactive Black 5 by T. versicolor strains MUM 94.04 and 04.100 reached 100 %. In addition, the highest white-rot fungi ligninolytic enzyme activities were found for these two strains.     

Biodecolourisation of some industrial dyes by white-rot fungi

Eight white-rot fungal strains were screened for biodecolourisation of eight dyes commercially employed in various industries. Decolourisation of Poly R 478 was used as a standard to ascertain the dye-decolourisation potential of various fungi. All the fungi tested significantly decolourised Poly R 478 on solid agar medium. When tested in a nitrogen-limited broth medium, Dichomitus squalens, Irpex flavus, Phlebia spp. and Polyporus sanguineus were better industrial dye decolourisers than Phanerochaete chrysosporium.     

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.     

The physiology of anthracene biodegradation by the white-rot fungus Bjerkandera sp. strain BOS55

A recently isolated white-rot strain, Bjerkandera sp. strain BOS55, displays high extracellular peroxidase activity, and rapidly degrades polycyclic aromatic hydrocarbons (PAH). In this study, the culture conditions for the biodegradation of the model PAH compound, anthracene, were optimized with respect to O₂, N, and C. An additional objective was to determine if the decolorization of the polymeric ligninolytic indicator dye, Poly R-478, could be correlated to anthracene biodegradation observed under a wide range of culture conditions. The supply of O₂ was found to be the most important parameter in the biodegradation of anthracene. Increasing culture aeration enhanced the biodegradation of anthracene and the accumulation of its peroxidase-mediated oxidation product anthraquinone. Decolorization of Poly R-478 was less affected by inadequate aeration. Provided that ample aeration was supplied, the degradation of anthracene under different culture conditions was strongly correlated with the ligninolytic activity as indicated by the rate of Poly R-478 decolorization. Concentrations up to 22 mM NH₄ ⁺ N did not repress anthracene biodegradation and only caused a 0%–40% repression of the Poly R-478 decolorizing activity in various experiments. A cosubstrate requirement of 100 mg glucose / mg anthracene biodegraded was observed in this study.     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Utilisation of lignocellulosic wastes for lignin peroxidase production by semi-solid-state cultures of Phanerochaete chrysosporium

In the present work, the production of ligninolytic enzymes by semi-solid-statecultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725),employing different lignocellulosic wastes as support, was investigated. Thewaste materials employed were grape seeds, wheat straw and wood shavings.Maximum lignin peroxidase activities of 1620 ± 123 U/l, 364 ± 35 U/l and 571 ± 42 U/l were attained, respectively. Nevertheless, lowmanganese-dependent peroxidase activities were found, being insignificantin the grape seed cultures. Moreover, the in vivo decolourisation of a model dye compound, the polymeric dye Poly R-478 (polyvinylamine sulfonateanthrapyridone), by the above-mentioned cultures was monitored to assessthe degrading capability of the extracellular liquid secreted by such cultures.The percentage of biological decolourisation attained by grape seed and woodshaving cultures was around 74% and 63%, respectively, whereas it was ratherlow (40%) in the wheat straw ones.     

Hydrogen Peroxide Production as a Limiting Factor in Xenobiotic Compound Oxidation by Nitrogen-Sufficient Cultures of Bjerkandera sp. Strain BOS55 Overproducing Peroxidases

The overproduction of ligninolytic peroxidase by the N-deregulated white rot fungus Bjerkandera sp. strain BOS55 under nitrogen-sufficient conditions had no noteworthy effect on the oxidation of anthracene or the decolorization of the polymeric aromatic dye Poly R-478 in 6-day-old cultures. Only when the endogenous production of H(inf2)O(inf2) was increased by the addition of extra oxygen and glucose could a 2.5-fold increase in the anthracene oxidation rate and a 6-fold increase in the Poly R-478 decolorization rate be observed in high-N cultures with 10- to 35-fold higher peroxidase activities than N-limited cultures. Further increase of the H(inf2)O(inf2) generation rate in high-N cultures with glucose oxidase led to an additional 3.5-fold increase in the anthracene oxidation rate (350 mg liter(sup-1) day(sup-1)) and a 10-fold increase in the Poly R-478 decolorization rate. These results indicate that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H(inf2)O(inf2). The absence of Mn, which decreased the manganese peroxidase titers and increased the lignin peroxidase titers, was associated with up to 95% improvements in the anthracene oxidation rate. The simultaneous presence of Mn and veratryl alcohol was observed to have a synergistic negative effect on the oxidation of anthracene and the decolorization of Poly R-478.