Anna Jarosz-Wilkołazka

Fungi and their ability to decolourize azo and anthraquinonic dyes

Abstract One hundred and fifteen fungi from different physio-ecological groups were compared for their capacity to decolourize two structurally different dyes in agar plates. We found that the azo dye, Acid Red 183, was much more resistant to decolourization by the examined strains in both solid and liquid cultures. Among the tested fungi, 69 strains showed decolourization of the anthraquinonic dye, Basic Blue 22, within 5–14 days, and only 16 strains were able to decolourize the azo dye, Acid Red 183, within 21 days. Furthermore, the potential of selected strains for decolourization of dyes was examined with regard to their extracellular oxidative factors both enzymatic and non-enzymatic. In static aqueous culture, the three selected fungi ( Bjerkandera fumosa , Kuehneromyces mutabilis , and Stropharia rugoso-annulata ) formed fungal mats, which did not decolourize any dye beyond some mycelial sorption. In comparison to the static cultures, the agitated cultures (180 rpm) removed 75 to 100% of the colour of Basic Blue 22 and 20 to 100% of Acid Red 183 colour.

Amperometric detection of mono- and diphenols at Cerrena unicolor laccase-modified graphite electrode: correlation between sensitivity and substrate structure

Graphite electrode modified with laccase from Cerrena unicolor served as a biosensor for detection of 30 phenolic compounds with different structures. Some correlations of the sensor response to the structures of substrates are discussed. This biosensor responded to: (i) nanomolar concentrations of some of the selected phenolic compounds, e.g., 2,6-dimethoxyphenol, coniferyl alcohol, caffeic acid, DOPAC and hydroquinone, (ii) micromolar concentrations, e.g., ferulic acid, syringic acid, dopamine, 3,4-dihydroxybenzoic acid and dl-noradrenaline, and (iii) millimolar concentrations in the case of phenol and 4-hydroxybenzaldehyde. Among the ortho- or para-substituted phenols, the sensitivity of the C. unicolor laccase-modified electrode increased in the following order H, CH(3), OH, OCH(3) and NH(3)(+) but in the case of para-substituted phenols, the K(m)(app) values were lower. The sensitivity of the laccase electrode increased with an additional OH group in para-substituted phenols. In the case of the selected compounds, kinetic data from electrochemical flow injection system were compared with those obtained from experiments in solution.

OXALATE PRODUCTION BY WOOD-ROTTING FUNGI GROWING IN TOXIC METAL-AMENDED MEDIUM

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.

Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of cd (II) ions on the activity of laccase.

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white‐rotting Basidiomycetes, were examined. Cd (II) ions were added to 10‐day‐old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy‐independent surface binding phenomenon, at the rate of ∼2mg Cd per g mycelial dry weight. An additional slower and energy‐dependent transport mechanism was also present, taking in ∼0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10‐day‐old cultures.

New Bioactive Fungal Molecules with High Antioxidant and Antimicrobial Capacity Isolated from Cerrena unicolor Idiophasic Cultures

Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39–90% for (a), 20–90% for (b), and 10–59% for (c) at the concentration of 6.25–800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25–800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu2O, Al2O3, ZnO, CuFe2O4Zn, CdO, and MnO2) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu2O- and ZnO-amended plates was observed. ZnO, CdO and Cu2O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu2O, MnO2, ZnO and CuFe2O4Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu2O, ZnO and MnO2. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.

Whole-cell fungal transformation of precursors into dyes

BackgroundChemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes.ResultsAn agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water.ConclusionsThis study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation.

Effect of Low Doses of Guaiacol and Ethanol on Enzymatic Activity of Fungal Cultures

The influence of low doses of guaiacol and ethanol, the natural effectors of lignin and phenolics transformations, on laccase and peroxidase activities produced by two strains of Basidiomycetes, Pleurotus sajor-caju and Trametes versicolor, was evaluated. Fungal mycelia were grown for 2 weeks on liquid media containing serial dilutions of guaiacol or ethanol ranging from 100−1 to 100−20 mol/L. Laccase and peroxidase activities in the medium were measured at the end of 2 weeks. The effect of low doses of guaiacol and ethanol on enzyme activities was manifested in an oscillating manner. Similar response patterns were observed when pure enzymes were exposed to the same serial dilutions of guaiacol and ethanol. T. versicolor cultures enriched with 40 mmol guaiacol (simulating natural environmental conditions) also displayed oscillating enzyme activity patterns in response to serial dilutions of guaiacol, but the maximum enzyme activity values were increased compared to those observed in cultures not receiving 40 mmol guaiacol. The differences between maxima and minima varied among the experimental groups and depended on the species of fungus, type of effector, and kind of enzyme. The results suggest the possibility of subtle regulation of enzymatic activity on the molecular level.

Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Versatile peroxidase of Bjerkandera fumosa: substrate and inhibitor specificity.

The inhibitor and substrate specificities of versatile peroxidase from Bjerkandera fumosa (VPBF) were studied. Two different effects were found: NaN(3), Tween-80, anthracene, and fluorene decreased the activity of VPBF, but p-aminobenzoic acid increased it. A mixed mechanism of effector influence on the activity of this enzyme was shown. The catalytic properties of VPBF in the oxidation of mono- and polycyclic aromatic compounds were studied also. 2,7-Diaminofluorene, ABTS, veratryl alcohol, and syringaldazine can be oxidized by VPBF in two ways: either directly by the enzyme or by diffusible chelated Mn(3+) as an oxidizing agent. During VPBF oxidation of 2,7-diaminofluorene, both with and without Mn(2+), biphasic kinetics with apparent saturation in both micromolar and millimolar ranges were obtained. In the case of ABTS, inhibition of VPBF activity by an excess of substrate was observed. Direct oxidation of p-aminobenzoic acid by versatile peroxidase was found for the first time. The oxidation of three- and four-ring PAHs by VPBF was investigated, and the oxidation of anthracene, phenanthrene, fluorene, pyrene, chrysene, and fluoranthene was shown. The products of PAH oxidation (9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone) catalyzed by VPBF were identified.