White Rot Fungi

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

Application of fungal-based bioaugmentation was evaluated for the remediation of creosote-contaminated soil at a wood-preserving site in West Virginia. Soil at the site contained creosote-range polycyclic aromatic hydrocarbons (PAHs) at concentrations in some areas that exceed industrial risk-based levels. Two white-rot fungi (Pleurotus ostreatus and Irpex lacteus) were evaluated for remediation effectiveness in a two-month bench-scale treatability test. Both fungi produced similar results, with up to 67.3 percent degradation of total PAHs in 56 days. Pilot-scale testing was performed at the site using Pleurotus ostreatus grown on two local substrate mixtures. During the 276-day field trial, total PAHs were degraded by up to 93.2 percent, with all individual PAHs except one achieving industrial risk-based concentrations. It was recommended that fungal-based remediation be applied to all contaminated soil at the site. © 2002 Wiley Periodicals, Inc.

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l−1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l−1 and 99 mg l−1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l−1 and 79 mg l−1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg−1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.

In order to improve the digestibility and nutrient availability in rumen, wheat straw was subjected to solid state fermentation (SSF) with white-rot fungi (i.e. Pleurotus ostreatus and Trametes versicolor) and the fermented biomass (called myco-straw) was evaluated for biochemical, enzymatic and nutritional parameters. The fungal treatment after 30 days led to significant decrease (P < 0.05) in cell wall constituents viz, acid detergent fiber (ADF), neutral detergent fiber (NDF), hemicellulose, lignin and cellulose to the extent of 35.00, 38.88, 45.00, 37.48 and 37.86%, respectively in P. ostreatus fermented straw, while 30.04, 33.85, 39.90, 31.29 and 34.00%, respectively in T. versicolor fermented straw. However, maximum efficiency of fermentation in terms of low carbohydrate consumption per unit of lignin degradation, favoring cattle feed production was observed for P. ostreatus on the 10th day (17.12%) as compared with T. versicolor on the 30th day (16.91%). The myco-straw was found to contain significantly high (P < 0.05) crude protein (CP; 4.77% T. versicolor, 5.08% P. ostreatus) as compared to control straw (3.37%). Metabolizable energy (ME, MJ/kg DM), percent organic matter digestibility (OMD) and short chain fatty acids (SCFAs; mmol) production also increased considerably from control straw (4.40, 29.91 and 0.292) to a maximum up to P. ostreatus fermented straw (4.92, 33.39 and 0.376 on 20th day) and T. versicolor fermented straw (4.66, 31.74 and 0.334 on 10th day), respectively. Moreover, the myco-straw had lower organic carbon and was rich in nitrogen with lower C/N ratio as compared to control wheat straw. Results suggest that the fungal fermentation of wheat straw effectively improved CP content, OM digestibility, SCFAs production, ME value and simultaneously lowered the C/N ratio, thus showing potential for bioconversion of lignin rich wheat straw into high energy cattle feed.

The culture conditions for extracellular secretion of lignin peroxidase by Pleurotus sajor caju MTCC–141 in the liquid culture growth medium amended with lignin containing natural substrates have been studied. Secretion of lignin peroxidase has been found to be maximum in the presence of bagasse. Lignin peroxidase from the liquid culture filtrate has been purified to homogeneity. Two isozymes having relative molecular masses 38 and 40 kDa have been isolated. The enzymatic characteristics like Km, pH, and temperature optima of the major isozyme (40 KDa) has been determined using veratryl alcohol, n-propanol, and H2O2 as the substrate. The Km values for veratryl alcohol, n-propanol, and H2O2 have been found to be 57 μ M, 500 μ M, and 80 μ M, respectively. The pH and temperature optima of lignin peroxidase have been found to be 3 and 30°C, respectively. The inhibition of the enzyme activity by sodium azide has been studied and it has been found to be uncompetitive, with KI value of 4 mM.

Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to homogeneity using Amicon concentration and DEAE cellulose chromatography. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis has been found to be 40 kDa. The Km values for veratryl alcohol and H2O2 for the purified enzyme were 58 and 83 μM, respectively. The calculated kcat value of the purified enzyme using veratryl alcohol as the substrate was 2.5 s−1. The pH and temperature optima of lignin peroxidase have been found to be 2.6 and 24°C, respectively.

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g−1 dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O2 enrichment. Furthermore, protease activity levels up to 34,000 U g−1 dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.

Four white-rot fungi isolated in Pakistan were used for decolorization of widely used reactive textile dyestuffs. Phanerochaete chrysosporium, Coriolus versicolor, Ganoderma lucidum and Pleurotus ostreatus were grown in defined nutrient media for decolorization of Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR for 10 days in shake flasks. Samples were removed every day, centrifuged and the absorbances of the supernatants were read to determine percentage decolorization. It was observed that P. chrysosporium and C. versicolor could effectively decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR. Drimarene Orange K-GL was completely decolorized (0.2 g/l after 8 days) only by P.chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also showed good decolorization efficiencies (0.19–0.2 g/l) on all dyes except Remazol Brilliant Yellow (0.07 g/l after 10 days). G. lucidum did not decolorize any of the dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 8 days).

Synthetic dyes are released in the effluent from a wide variety of industries such as textile, tannery, packed food, pulp and paper and paint, thus threatening various forms of life. Bioremediation is always considered as cost effective and eco-friendly way for the treatment of recalcitrant dyes and effluents. Non-white rot fungus Aspergillus fumigatus A23 and white rot fungus Phanerochaete chrysosporium were used for comparative study of decolorization of individual dyes and simulated textile effluent (STE). Both the fungi could effectively decolorize STE under optimized conditions of medium (potato dextrose agar medium), temperature (40ºC for A. fumigatus A23 and 30ºC P. chrysosporium), pH (4.0 for A. fumigatus A23 and 5.0 for P. chrysosporium) and agitation (100 rpm for A. fumigatus A23 and P. chrysosporium). The decolorization of STE by A. fumigatus A23 and P. chrysosporium was 86% and 62% respectively after 7d incubation. The key mechanisms involved in dye removal by the fungus appeared to be adsorption and absorption and the biotransformation occurred only after absorption of the dye. Analysis of samples before and after treatment with fungus using TLC indicated the biotransformation of dye.