Olive mill wastewater biodegradation potential of white-rot fungi--Mode of action of fungal culture extracts and effects of ligninolytic enzymes.

Forty-nine white-rot strains belonging to 38 species of Basidiomycota were evaluated for olive-mill wastewater (OMW) degradation. Almost all fungi caused high total phenolics (>60%) and color (⩽ 70%) reduction, while COD and phytotoxicity decreased to a lesser extent. Culture extracts from selected Agrocybe cylindracea, Inonotus andersonii, Pleurotus ostreatus and Trametes versicolor strains showed non-altered physicochemical and enzymatic activity profiles when applied to raw OMW in the presence or absence of commercial catalase, indicating no interaction of the latter with fungal enzymes and no competition for H2O2. Hydrogen peroxide's addition resulted in drastic OMW's decolorization, with no effect on phenolic content, suggesting that oxidation affects colored components, but not necessarily phenolics. When fungal extracts were heat-treated, no phenolics decrease was observed demonstrating thus their enzymatic rather than physicochemical oxidation. Laccases added to OMW were reversibly inhibited by the effluent's high phenolic load, while peroxidases were stable and active during the entire process.

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

Influence of iron and copper nanoparticle powder on the production of lignocellulose degrading enzymes in the fungus Trametes versicolor.

White rot fungi are one of the key group of microorganisms that help to enrich the soil via degradation of wood. In the current communication, influence of iron and copper nanoparticles on the production of lignocellulolytic enzymes by Trametes versicolor have been investigated. The production of enzymes in the presence of the two nanoparticles was compared to that of ferrous and cupric ions respectively. Results show that both the tested nanoparticles alter the production profile of the lignocellulolytic enzymes when compared to the control set. The production of laccase was not influenced by iron nanoparticles but was effected by copper nanoparticles within 24h of incubation. Both the nanoparticles decreased the production of beta-glucosidase, beta-xylosidase and cellobiohydrolase significantly. However, the production profile of Mn-peroxidase and remained statistically similar to that of control when the organism was incubated with iron and copper nanoparticles. The production profiles were also different when one compares the ionic form of metals and the nanoparticles, suggesting different mechanism of action of the particles on the organism. The difference in the production profile was not growth related as no significant difference was recorded for either form of iron and copper on the growth of T. versicolor.

Synthesis of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide and the oxidation of polycyclic aromatic hydrocarbons.

Chelating sorbents with diethylenetriaminepenta(methylene-phosphonic acid) (DTPMPA) and ethylenediaminetetraacetic acid ligands immobilized on zirconia matrix were prepared and subsequently saturated with Cu(II). All the Cu chelates catalyzed decomposition of H(2)O(2) yielding highly reactive hydroxyl radicals. All of them were also able to catalyze degradation of polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene and benzo[b]fluoranthene). The most effective DTPMPA-based catalysts G-32 and G-35 (10 mg ml(-1) with 100 mmol H(2)O(2)) caused almost complete decomposition of 15 ppm anthracene and benzo[a]pyrene during a five day catalytic cycle at 30 degrees C. Anthracene-1,4-dione was the main product of anthracene oxidation by all catalysts. The catalysts were active in several cycles without regeneration.

Decolorization of Orange G and Remazol Brilliant Blue R by the white rot fungus Dichomitus squalens: toxicological evaluation and morphological study.

Dichomitus squalens efficiently decolorized both Orange G and Remazol Brilliant Blue R (RBBR) at concentrations of 0.5gl(-1) and 3gl(-1) in static and shaken culture and also on solid medium within 14d. The presence of the dyes in the culture medium mostly caused a decrease in biomass production and in growth rate, which was more significant in the case of RBBR. After 14d of cultivation, electron microscopy showed substantial morphological changes in mycelia of D. squalens growing in media containing dyes. The hyphae deformations were more intensively manifested in solid media than in liquid culture. In all the cases, the morphological changes were more prominent in the presence of RBBR. Higher concentrations of both dyes brought about more intensive changes. The toxicity of synthetic dyes Orange G and RBBR was tested using a bioassay based on the growth inhibition of duckweed Lemna minor. Two endpoints such as the number of fronds and their weight were studied during the bioassay. The results showed higher toxicity of RBBR than that of Orange G. The toxicity of both dyes decreased after the decolorization process.

Basidiomycete cultures on perlite survive successfully repeated freezing and thawing in cryovials without subculturing.

Mycelial basidiomycete cultures on perlite in cryovials survived successfully three successive cycles of freezing, storage in liquid nitrogen (LN) and thawing without noticeable changes. This indicates that using perlite as a carrier for cryopreservation could in most cases overcome difficulties caused by interrupted supply of LN or electric power during the storage. Cultures on perlite can also be reused for successive inoculations.

The influence of extracellular H2O2 production on decolorization ability in fungi.

A set of 50 randomly chosen fungal strains belonging to different basidiomycete species was tested for H2O2 and ligninolytic enzyme production and for decolorization of synthetic dyes Orange G and Remazol Brilliant Blue R. The decolorization capacity of individual strains was influenced by the level of H2O2 and laccase activity. The strains producing H2O2 at a concentration of 1.0-1.5 microM exhibited the most efficient decolorization; higher or lower H2O2 concentration reduced this ability. None of the strains without a detectable laccase activity was able to decolorize the tested dyes.

Basidiomycete cryopreservation on perlite: evaluation of a new method.

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.

Influence of dimethyl sulfoxide on extracellular enzyme production by Pleurotus ostreatus.

Dimethyl sulfoxide (DMSO) is commonly used as a co-solvent to dissolve poorly water-soluble biologically active agents to assess their biological activities such as for enzyme induction. The question addressed was whether DMSO can be assumed to be an inert co-solvent. The influence of DMSO on the production of extracellular enzymes by Pleurotus ostreatus was investigated. DMSO functioned as either an inducer or a repressor, depending on the enzyme studied. The production of laccase and endo-1,4-beta-xylanase increased by 29 and 250%, respectively, in presence of DMSO. However, DMSO repressed the activities of manganese peroxidase, beta-glucosidase, beta-xylanase, and endo-1,4-beta-glucanase by 30, 33, 99 and 16%, respectively. These results raise concerns about the interpretation of bioactivity measurements when DMSO is assumed to function as an inert co-solvent to solubilize water-insoluble molecules.

Synthetic dye decolorization capacity of white rot fungus Dichomitus squalens.

The ability to decolorize eight chemically different synthetic dyes (Orange G, Amaranth, Orange I, Remazol Brilliant Blue R (RBBR), Cu-phthalocyanin, Poly R-478, Malachite Green and Crystal Violet) by the white rot fungus Dichomitus squalens was evaluated on agar plates. The fungus showed high decolorization capacity and was able to decolorize all dyes tested, but not to the same extent. Some of the dyes did not limit the decolorization capacity of the strain tested even at a concentration of 2g/l. The presence of the dyes in solid media reduced the mycelial growth rate of D. squalens; a positive correlation was found between the growth rate and the decolorization ability. Decolorization of Orange G and RBBR was studied also in liquid culture, where both dyes caused an enhancement of ligninolytic enzyme and overall hydrogen peroxide production and a decrease of biomass production. RBBR was removed to a higher extent than Orange G.

Orange G and Remazol Brilliant Blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus.

Thirty different white rot strains were screened for Orange G and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected on the basis of this screening, were used for decolorization study in liquid media. All three strains efficiently decolorized both Orange G and RBBR, but they differed in decolorization capacity depending on cultivation conditions and ligninolytic enzyme production. Two different decolorization patterns were found in these strains: Orange G decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and MnP cooperated in the decolorization processes.

Copper-ligand complex for the decolorization of synthetic dyes.

The reaction system containing Cu(II), hydrogen peroxide and D-arabinono-1,4-lactone was found to be effective in the decolorization and reduction of toxicity of azo, thiazine-, triphenylmethane- and anthraquinone-based synthetic dyes. More than 85% decolorization was obtained with 100ppm Acridine Orange, Azure B, Chicago Sky Blue 6B, Crystal Violet, Evans Blue, Poly B-411, Reactive Blue 2, Reactive Blue 5, and Remazol Brilliant Blue R incubated for 24h in the presence of 10mM CuSO(4), 20mM D-arabinono-1,4-lactone and 80 mM H(2)O(2). The rate of decolorization was not affected by pH in the range of 3-9. The rapid decolorization was accompanied by a fast decomposition of H(2)O(2) in the reaction mixture and by a fast production of hydroxyl radicals.

Decolorization of synthetic dyes using a copper complex with glucaric acid.

Selected azo, acridine, triphenyl methane, anthraquinone and thiazine-based dyes were decolorized using a catalytic system consisting of Cu(II)/glucaric acid/H(2)O(2). More than 90% decolorization was obtained with 100 ppm Acridine Orange, Azure B, Chicago Sky Blue, Crystal Violet, Methyl Orange, Poly B-411, Reactive Black 5, Reactive Blue 2, and Remazol Brilliant Blue R within 24 h. Seventy to eighty percent decolorization was achieved within the first 6 h. The decolorizaton was not affected by pH. The involvement of hydroxyl radicals produced in the system in the decolorization of the dye molecules was confirmed by electron spin resonance study.

Decolorization of structurally different synthetic dyes using cobalt(II)/ascorbic acid/hydrogen peroxide system.

The cobalt(II)/ascorbic acid/hydrogen peroxide system was used for decolorization of azo, acridine, anthraquinone, thiazine and triphenylmethane dyes. More than 90% decolorization was obtained with all dyes except Remazol Brilliant Blue R (75%). With other transition metals the system was less efficient. With copper, higher concentration and prolonged incubation time was necessary to obtain the same extent of decolorization. The rate of decolorizaton was not affected by pH in the range of 3-9. The reaction is very fast, with more than 90% decolorization being attained within 15 min. The system produces hydroxyl radicals which are responsible for the decolorization.

Lignin degrading system of white-rot fungi and its exploitation for dye decolorization.

With global attention and research now focused on looking for the abatement of pollution, white-rot fungi is one of the hopes of the future. The lignin-degrading ability of these fungi have been the focus of attention for many years and have been exploited for a wide array of human benefits. This review highlights the various enzymes produced by white-rot fungi for lignin degradation, namely laccases, peroxidases, aryl alcohol oxidase, glyoxal oxidase, and pyranose oxidase. Also discussed are the various radicals and low molecular weight compounds that are being produced by white-rot fungi and its role in lignin degradation. A brief summary on the developments in research of decolorization of dyes using white-rot fungi has been made.