White Rot Fungi

Biological upgrading of wheat straw by the white-rot fungi Phanerochaete chrysosporium, Pleurotus eryngii, Phlebia radiata, and Ceriporiopsis subvermispora was monitored during 60-day solid-state fermentation. Analysis of straw included determination of weight loss and lignin content, color analysis, and infrared spectroscopy, whereas the studies on the water-soluble fractions were carried out by infrared spectroscopy, elementary analyses and quantification of the total phenols and reducing sugars. The most selective degradation of lignin was produced by P. eryngii and especially by C. subvermispora, the former species releasing the greatest amount of colored water-soluble products, whereas an increase in straw brightness was caused by C. subvermispora. In general, the composition of the water-soluble fraction correlated with the extent of straw transformation. The initial fermentation stage (0-15 days) was characterized by the accumulation of water-soluble products from lignin degradation and fungal metabolism, the concentration of which tended to stabilize in the second stage (16-60 days). The degree of delignification at the second stage tended to coincide with the decrease of the water-soluble nitrogen.

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.

The laccase gene lac48424-1 and its corresponding full-length cDNA were cloned and characterized from a novel white-rot fungi Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes. The 1563 bp full-length cDNA of lac48424-1 encoded a mature laccase protein containing 499 amino acids preceded by a signal peptide of 21 amino acids. The deduced protein sequence of LAC48424-1 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. The functionality of lac48424-1 gene encoding active laccase was verified by expressing the gene in the yeast Pichia pastoris successfully. It was found that the recombinant laccase produced by the yeast transformant could decolorize different dyes. The 5 0 -flanking sequence upstream of start codon was obtained by Self-Formed Adaptor PCR. Many putative cis-acting responsive elements involved in the transcriptional regulation were identified in the promoter region of lac48424-1.

Six strains of white-rot fungi isolated from southern Chile were evaluated for their ergosterol/biomass correlation and ligninolytic potential in solid medium to formulate pellets for Reactive Orange 165 (RO165) decolourization. The fungus Anthracophyllum discolor was selected to formulate complex pellets (fungal mycelium, sawdust, and activated carbon), coated pellets (complex pellet + alginate) and simple pellets (fungal mycelium). The activity of ligninolytic enzymes (laccase, manganese peroxidase, manganese-independent peroxidase, and lignin peroxidase) was evaluated in both the complex and coated pellets in modified Kirk medium, and the morphology of the pellets was studied using scanning electron microscopy (SEM). Complex pellets of A. discolor showed a higher enzymatic production mainly MnP (38 U L-1 at day 15) compared to coated and simple pellets. Examinations using SEM showed that both pellets produced a black core that was entrapped by a layer of fungal mycelium. Decolourization of RO165 was demonstrated with all the pellets formulated. However, the highest and fastest decolourization was obtained with complex pellets (100% at day 8). Therefore, complex pellets of A. discolor can be used for the biological treatment of wastewater contaminated with RO165.

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.

Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h.

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized.

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 control of water pollution has become of increasing importance in recent years. The release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. Tightening government legislation is forcing textile industries to treat their waste euent to an increasingly high standard. Currently, removal of dyes from euents is by physio-chemical means. Such methods are often very costly and although the dyes are removed, accumulation of concentrated sludge creates a disposal problem. There is a need to ®nd alternative treatments that are eective in removing dyes from large volumes of euents and are low in cost, such as biological or combination systems. This article reviews the current available technologies and suggests an eective, cheaper alternative for dye removal and decolourisation applicable on large scale. Ó

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 is amorphous in nature, lacks stereoregularity, and is not susceptible to hydrolytic attack. Despite its resistant nature, it is however degraded by various microorganisms, particularly, white-rot fungi. Such fungi are capable of extracellular production of lignin peroxidase, manganese peroxidase, and laccase, the three major enzymes associated with ligninolysis. Though all white-rot fungi do not produce all the three enzymes, laccase occupies an important place in ligninolysis. Laccase belongs to a diverse group of enzymes called oxidoreductases and is also known as benzenediol: oxygen oxidoreductase. They have low substrate specificity. The copper-containing enzyme laccase has been detected in a variety of organisms such as bacteria, fungi, plants, and insects. Mostly, these are extracellular proteins, although intracellular laccases have also been detected in some fungi and insects. Fungal laccases are believed to play a variety of roles, such as, morphogenesis, pathogenesis, and lignin degradation. As an oxidase, laccase is used in many agricultural, industrial, and medicinal applications. Current investigations are focused on laccase-based biooxidation, biotransformation, biosensor, and enzymatic synthesis of organic compounds. By enhancing laccase production using different physiochemical parameters, better understanding of the mechanism for the reactions of interest, and optimizing the catalytic activity of laccase, it can be used in a better way in diverse fields of biotechnology.

White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. This ligninolytic system of white-rot fungi (WRF) is directly involved in the degradation of various xenobiotic compounds and dyes. This review summarizes the state of the art in the research and prospective use of WRF and their enzymes (lignin-modifying enzymes, LME) for the treatment of industrial effluents, particularly dye containing effluents. The textile industry, by far the most avid user of synthetic dyes, is in need of ecoefficient solutions for its colored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the biocatalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes. D

The ligninolytic enzymes of white-rot fungi have a broad substrate specificity and have been implicated in the transformation and mineralization of organopollutants with structural similarities to lignin. This review presents evidence for the involvement of these enzymes in white-rot fungal degradation of munitions waste, pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, bleach plant effluent, synthetic dyes, synthetic polymers, and wood preservatives. Factors relating to the feasibility of using white-rot fungi in bioremediation treatments for organopollutants are discussed.

Lignocellulosic waste represents huge amounts of unutilized renewable resource. The use of the polysaccharides in the lignocellulosic complex is limited due to their high lignin content. White rot fungi are capable of selectively degrading lignin, thereby upgrading it. The focus of this article is on the potential utilization of edible mushrooms of the genus Pleurotus, via solid state fermentation, using cotton plant stalks as a substrate. This material poses agrotechnical problems since the stalks have a fibrous structure similar to that of hardwood. Potential uses for this material are as a fuel in rural areas, a substrate for mushrooms, an animal feed and substrate for paper making. In this study, degradation of cotton stalks by Pleurotus is described using chemical analyses and scanning electron microscopy. During four weeks of solid state fermentation, lignin content significantly decreased and in vitro digestibility was increased. The fermentation product was consumed by ruminants at a level of up to 40% of their diet.

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.

In order to decolourise olive oil mill wastewaters (OOMW) efficiently, production and differential induction of ligninolytic enzymes by the white rot Coriolopsis polyzona, were studied by varying growth media composition and/or inducer addition. Among various possible inducers, veratryl alcohol appeared to be the most efficient to enhance specific productions of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase by a factor of 18.5, 20.8 and 55, respectively. Ligninolytic enzymes were better produced in glucose based medium with a low nitrogen level (2.2 mM) under O 2 atmosphere. The addition of 5 mM veratryl alcohol resulted in a maximal production of LiP, whereas maximal MnP and laccase were obtained at 10 mM. LiP production was totally repressed in presence of 100 lM Mn 2+ . The extrapolation of these conditions on OOMW based media was carried out at different effluent dilutions and the possible role of the different ligninolytic enzymes in OOMW decolourisation was studied. A better effluent decolourisation was obtained under LiP induction condition (5 mM veratryl alcohol) than when LiP was repressed (100 lM Mn 2+ ). Furthermore, high levels of laccase had a detrimental effect on OOMW decolourisation concomitant to the formation of soluble polymeric aromatic compounds.

This review discusses the applications of laccase in the forest products industry. Laccases are versatile, phenol-oxidizing enzymes obtained from white-rot fungi and other natural sources; when acting on lignin, they can display both ligninolytic and polymerizing (cross-linking) abilities. The substrate range of laccase can be extended to cover both phenolic and non-phenolic compounds by means of laccase-mediator systems (LMS), which makes laccase suitable for, e.g., biobleaching of lignocellulosic pulps. As laccase/LMS can also help remove pitch, phenolic contaminants, and dyes from wood-based materials and water, laccase technology is applicable to virtually the entire production chain of paper products from pulping to recovery of secondary fibers and effluent treatment. Indeed, most of the published research and applications of laccase/LMS in the forest products industry relate to the pulp and paper sector, where particular emphasis has been placed on studying laccase/LMS for use in biobleaching and mill water treatment. Emerging research areas include the tailoring of lignocellulosic materials by laccase-assisted biografting of phenols and other compounds, and the use of laccase for adhesion enhancement in binderless wood boards.

Trametes trogii BAFC 463 culture fluids (containing 110 U ml À1 laccase; 0.94 U ml À1 manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu þ2 , Pb þ2 or Cd þ2 ) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.

The rate and efficiency of decolorization of poly R-478-or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 mg g − 1 ) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28°C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.

Treatment of effluents from dye-based industries poses a major problem and biotreatment with white rot fungi seems to be a viable option. In this study, Phanerochaete chrysosporium, a commonly used white rot fungus, was used to biodegrade several synthetic dyes of varying structures, namely azo, anthraquinone, thiazine and vat dyes. The decolorization potential of P. chrysosporium for seven dyes namely, Methyl violet, Congo red, Acid orange, Acid red 114, Vat magenta, Methylene blue and Acid green was studied. The effect of various operational parameters, namely dyes concentration (20-400 mg/l), pH (2-7), temperature (20-45 8C) and inoculum size (0.25-4 ml) on the maximum percentage decolorization were investigated. Studies were carried out using free cells and fungal cell entrapped calcium alginate beads of different sizes. The kinetics parameters 'K dye ' and 'V dye max ' for the decolorization process for all the seven dyes were estimated through Lineweaver-Burk plots. #

Many synthetic dyes present in industrial wastewaters are resistant to degradation by conventional treatments. Decolorization of four synthetic azo dyes was examined in two white rot fungal cultures. In solidified culture medium, Phanerochaete chrysosporium partially decolorized all the dyes tested, while Pleurotus sajorcaju totally decolorized amaranth, new coccine, and orange G, but not tartrazine. In liquid culture medium, P. chrysosporium totally decolorized amaranth, new coccine and orange G, and 60% tartrazine. Pleurotus sajorcaju totally decolorized amaranth and new coccine, 50% orange G and a maximum of 20% tartrazine. Neither fungus showed lignin peroxidase or veratryl alcohol oxidase activities, suggesting that these enzymes may not be involved in the decolorization. Manganese-peroxidase and ␤-glucosidase may be involved in the decolorization of the dyes by P. chrysosporium, whereas in P. sajorcaju a laccase active toward o-dianisidine, and glucose-1-oxidase might participate in the process.

We are studying the enzymatic modification of polycyclic aromatic hydrocarbons ( PAHs ) by the laccase from Coriolopsis gallica UAMH 8260. The enzyme was produced during growth in a stirred tank reactor to 15 units ml -1 , among the highest levels described for a wild -type fungus; the enzyme was the major protein produced under these conditions. After purification, it exhibited characteristics typical of a white rot fungal laccase. Fifteen azo and phenolic compounds at 1 mM concentration were tested as mediators in the laccase oxidation of anthracene. Higher anthracene oxidation was obtained with the mediator combination of ABTS and HBT, showing a correlation between the oxidation rate and the mediator concentration. Reactions with substituted phenols and anilines, conventional laccase substrates, and PAHs were compared using the native laccase and enzyme preparations chemically modified with 5000 MW -poly( ethylene glycol ). Chemically modified laccase oxidized a similar range of substituted phenols as the native enzyme but with a higher catalytic efficiency. The k cat increase by the chemical modification may be as great as 1300 times for syringaldazine oxidation. No effect was found of chemical modification on mediated PAH oxidation. Both unmodified and PEG -modified laccases increased PAH oxidation up to 1000 times in the presence of radical mediators. Thus, a change of the protein surface improves the mediator oxidation efficiency, but does not affect nonenzymatic PAH oxidation by oxidized mediators.

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.

To investigate the growth and production of lignocellulosic enzymes from Lentinus tigrinus, solid state fermentation (SSF) during 110 days was performed by using wheat straw as substrate. The BE obtained was 62% and three flushes of mushrooms were harvested. L. tigrinus showed the capacity to degrade wheat straw, causing a 21.49% decrease in lignin content and a 53.26% decrease in cellulose. The water extractives (aromatic compounds and reducing sugars) increased to 121.44% along incubation time. All of the enzyme activities were high during colonization and ligninases declined drastically during fruit body formation. After harvesting the last flush, enzyme activities increased rapidly to highest levels, except for laccase activity. Cellulases showed the maximum of activity around 90 days post inoculation. Laccase titres reached a peak of 30 U g À1 at day 20 and MnP had two peaks of around 750 mU g À1 at days 20 and 90. Aromatic compounds from lignin degradation were not inducers of ligninases. Laccase was the most stable enzyme showing complete stability after 42 h at 40 8C. All enzymes showed acid optimum pH. The minor value was recorded for laccase and endoglucanase (pH 3.0).

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).

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.

The extracellular ligninolytic enzymatic activity of the fungus Irpex lacteus (Polyporus tulipiferae) grown in nonimmersed liquid culture under nitrogen limitation was characterized. Laccase (LAC), manganese-dependent lignin peroxidase (MNP) and lignin peroxidase (LIP) activities were detected and their formation followed a temporal sequence peaking at day 4, 7 and 11, respectively. Three LIP isozymes, designated LIP1, LIP2 and LIP3, were isolated from manganese-depleted cultures, and exhibited molecular masses of 41, 41 and 44 kDa, respectively, pIs of 5.0, 4.9 and <4.6, respectively, and to 4-5% glycosylation. All isozymes were active towards a range of phenolic substrates, with veratryl alcohol being the preferred one. The kinetics constants obtained for veratryl alcohol and H 2 O 2 were comparable to those of LIPs from other white-rot fungi. The N-terminal sequences of LIP1 and LIP2 were identical and presented 64% similarity with LIP3, and were closely related to the amino acid sequences of LIP isozymes from Phlebia radiata (LIG III), Trametes versicolor (LIG C) and Phanerochaete chrysosporium (GLG2, GLG3 and GLG4). Sequence alignment with other reported LIPs revealed fewer identical or similar residues. Taken together, these data suggest that despite similarity in catalytic properties, kinetics parameters and spectral characteristics, there seems to be high degree of diversity among LIPs of different white-rot fungi.

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.

The growth mechanisms of filamentous fungi in liquid cultures ranges from dispersed mycelia to compact pellets. The excessive growth of mycelia gives rise to practical and technical difficulties in culturing fungi and consequently the possibility to control and regulate hyphal extension and pellet size is of great importance for their potential application in continuous operation. Here we show how the control of fungal growth on the surface of a biofilm by means of a pulsing flow can enhance the efficiency and stability of processes with different objectives, i.e., the production of a ligninolytic enzyme, the decolourisation of synthetic dyes and the detoxification of coloured effluents.

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented. #

Today, more than 3,000 pharmaceutical and personal care products (PPCPs) are used and released into the environment at low doses but they are barely degraded in wastewater treatment plants. One of the potential alternatives to effectively degrade PPCPs is based on the use of white-rot fungi (WRF) and involves the oxidative action of extracellular fungal enzymes. The aim of this work is to study the potential ability of three WRF strains, an anamorph species of Bjerkandera sp. R1, Bjerkandera adusta and Phanerochaete chrysosporium, to degrade PPCPs belonging to different therapeutic groups: anti-depressants (citalopram and fluoxetine), antibiotics (sulfamethoxazole), anti-inflammatory drugs (diclofenac, ibuprofen and naproxen), anti-epileptics (carbamazepine), tranquilizers (diazepam) and fragrances (celestolide, galaxolide and tonalide). The results reported complete degradation of all the PPCPs except for fluoxetine and diazepam, which were partially removed in percentages from 23 to 57%. In the case of fragrances, these compounds were neither detected in the fungal cultures nor in the abiotic controls, indicating the possibility of volatilization during the experiment.

In this report, we have identified oxalic acid as an important metabolite elaborated in the response of wood-rotting fungi to toxic metal stress. The formation of oxalate crystals by white rot fungi (Bjerkandera fumosa, Phlebia radiata and Trametes versicolor) and the brown rot fungus Fomitopsis pinicola, grown on media containing high levels of toxic metal ions has been visualized using scanning electron microscopy (SEM) with energy-dispersive X-ray micro-analysis (EDXA) and HPLC. There were no significant differences between the growth of controls (metal-free) and on the 0.5% CaCO 3 , Co 3 (PO 4 ) 2 or Zn 3 (PO 4 ) 2 -amended plates. ZnO inhibited the growth of all strains. Crystals were not detected in Zn 3 (PO 4 ) 2 -amended plates. The four examined strains displayed the formation of crystals on ZnO, Co 3 (PO 4 ) 2 and CaCO 3 -amended plates.

Essential oils and their derivatives have a long history of safe usage as antimicrobial agents in food industry. In this study various essential oils and extracts from plants were screened for their ability to inhibit wood decay and termite attack in laboratory decay and termite resistance tests using treated wood specimens. In the laboratory decay resistance tests, wood specimens treated with essential oil compounds were subjected to brown-rot fungus, Tyromyces palustris, and white-rot fungus, Trametes versicolor for three months. The specimens were also subjected to termite attack by subterranean termites, Coptotermes formosanus for three weeks in laboratory conditions. The formulation with cinnamaldehyde (DF3) was found to be effective against both the brown-rot and white-rot fungi used in the study. However, the formulation with cinnamic acid (DF8) was able to protect wood specimens against only the white rot fungus and about 50% mass loss occurred in the specimens subjected to the brown-rot fungus. The mass losses in the wood specimens treated with cassia oil containing formulation (DF4) showed that cassia oil was effective against both fungi. The wood tar oil and dodecanal compounds also inhibited fungal decay in the specimens. The wood specimens treated with the formulations used in termite resistance tests were more resistant against the termites when compared to specimens treated with the formulations in decay resistance tests. Even after severe weathering process, treated wood specimens showed resistance against the subterranean termites. Results suggest that essential oils and plant extracts might be important to develop new wood preservatives that are less harmful to the environment and humans than recently available ones. Zusammenfassung In der Nahrungsmittelindustrie werden ätherische Öle und deren Derivate bereits seit langem als sichere antimikrobielle Mittel eingesetzt. In dieser Studie wurde in Laborprüfungen anhand behandelter Holzproben untersucht, inwieweit sich verschiedene ätherische Öle und Pflanzenextrakte zur Hemmung von Holzfäule und Termitenbefall eignen. In Laborprüfungen zur Fäuleresistenz wurden die Holzproben mit ätherischen Ölen behandelt und für drei Monate dem Braunfäulepilz Tyromyces palustris sowie dem Weißfäulepilz Trametes versicolor ausgesetzt. Die Holzproben wurden ebenfalls einem dreiwöchigen Termitenbefall unter Laborbedingungen ausgesetzt. Dabei wurden die Bodentermiten Coptotermes formosanus verwendet. Die Rezeptur mit Zimtaldehyd (DF3) erwies sich sowohl gegen Braun-als auch gegen Weißfäulepilze als wirksam. Dagegen bot die Rezeptur mit Zimtsäure (DF8) nur einen Schutz gegen Weißfäulepilze. Bei Braunfäulepilzen ausgesetzten Proben war ein Masseverlust von 50% zu verzeichnen. Die Masseverluste in den Holzproben, die mit Rezepturen, die Kassiaöl (DF4) enthielten, behandelt wurden, zeigten, dass Kassiaöl gegen beide Pilze wirksam ist. Holzteeröl und Dodecanal-Verbindungen hemmten ebenfalls den Pilzbefall in den Proben. Die Holzproben, die mit den in Termitenresistenzversuchen verwendeten Rezepturen behandelt wurden, zeigten sich gegen Termitenbefall resistenter als Proben, die mit den in Fäuleresistenzversuchen verwendeten Rezepturen behandelt wurden. Sogar nach starker Bewitterung erwiesen sich die behandelten Proben als resistent gegen Bodentermiten. Die Ergebnisse zeigen, dass ätherische Öle und Pflanzenextrakte bei der Entwicklung neuer Holzschutzmittel, die für die Umwelt und den Menschen weniger schädlich als die bereits erhältlichen Mittel sind, eine wichtige Rolle spielen können.

The ability of eight white rot fungi: Coriolopsis rigida, Coriolus versicolor var. antarcticus, Peniophora sp., Phanerochaete sordida, Pycnoporus sanguineus, Steccherinum sp., Trametes elegans and Trametes villosa to selectively delignify loblolly pine (Pinus taeda) chips was studied. They were selected among 34 basidiomycetes from Argentina because of their capacity to decolorize Poly R-478 and Azure B. Fungal pretreatment caused changes in wood chemical composition as well as in physical structure. The present study allowed the identification of a new strain, potentially a candidate for use in softwoods biopulping processes. Results showed that P. sanguineus was able to reduce lignin content in 11% in 14 days of treatment, but also that P. taeda wood suffered notable structural changes of lignin and hemicelluloses during the treatment, as revealed from 13 C CP-MAS NMR spectra. An increase of 15% in porosity of decayed wood confirmed physical changes due to fungal attack. #

Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486 U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725 U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.

10 Abstract 11 Control and hot water treated particles of cypress (Cupressus spp.) were used to manufacture cement-bonded particleboards 12 (CBPB). Three replications were carried out for each treatment, totaling six single layered boards. Physical and mechanical 13 properties of the boards were evaluated according to ASTM D 1037-96a [Annual Book of ASTM Standards, vols. 04-09. Phila-14 delphia: American Society for Testing and Materials--ASTM, ASTM D 1037-96a, 1998] standard. The wood of cypress showed 15 suitability as raw material for the manufacture of CBPB. All the properties of cypress CBPB were higher than the Bison HZ type 16 building boards used as reference. The light color and easy wood processing of cypress wood are favorable parameters for panels 17 manufacturing. The results corroborate those reported for CBPB using pine, rubberwood, acacia, babassu, and eucalyptus particles. 18 The CBPB were also submitted to fungi decay resistance test according to ASTM D 2017-81 [Annual Book of ASTM Standards, 19 vols. 04-09. Philadelphia: American Society for Testing and Materials--ASTM, 1994-e1. ASTM D 2017 standard and 20 exposed to the attack of Gloeophyllum trabeum (Persoon ex Fries) Murr. and Trametes versicolor (Linnaeus ex Fries) Pil at. Twelve 21 specimens were tested for each treatment and the average weight loss was determined after 12 weeks of exposure. Laboratory tests 22 showed that there was no measurable wood degradation (weight loss). Fungi mycelium did not even cover completely the surface of 23 the specimens. CBPB when exposed to both brown-and white-rot fungi, rather than weight loss, showed weight gains ranging from 24 2% to 4%. Therefore, CBPB is technically suitable for exterior use where both moisture and favorable conditions for fungi 25 development are present. 26

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.