Radioactive Contamination of Wild Mushrooms from Ukraine under Conditions of Contrasting Radiation Loads: 36 Years after the Chernobyl Nuclear Power Plant Catastrophe.

It has been 36 years since the Chernobyl Nuclear Power Plant catastrophe, but the consumption of wild mushrooms in Ukrainian Polissya is still dangerous since its territory is heavily contaminated by radionuclides. The aim of this study was to estimate 137Cs and 90Sr uptake in wild mushrooms from locations with contrast radioactive loads (Zhytomyr Polissya, Ukraine). In mushroom samples collected from Drevlyanskyi Nature Reserve since 2013 (average levels of soil surface contamination with 137Cs are 555-1480 kBq m-2), the highest levels of 137Cs were observed in symbiotroph species - Imleria badia (≤ 2680 kBq kg-1 dry mass [dm]), Tricholoma equestre (≤ 1420 kBq kg-1 dm), Lactarius rufus (≤ 602 kBq kg-1 dm), Sarcodon imbricatus (≤ 464 kBq kg-1 dm), Leccinum scabrum (≤ 117 kBq kg-1 dm), Suillus bovinus (≤ 118 kBq kg-1 dm), and Boletus edulis (≤ 96 kBq kg-1 dm). 90Sr activity was significantly lower, with the highest levels detected in Russula emetica (193 Bq kg-1 dm), Daedaleopsis confragosa (145 Bq kg-1 dm), and Hypholoma fasciculare (141 Bq kg-1 dm). The 137Cs/90Sr ratio in fruiting bodies in samples ranged from 6.1 (Bovistella utriformis) to 28,979 (T. equestre). Activity concentrations in mushrooms from locations with relatively low contamination with 137Cs (18.5-27.75 kBq m-2) also reached the highest values in symbiotroph species I. badia (7698 Bq kg-1 dm), Lactarius vellereus (6072 Bq kg-1 dm), and S. luteus (1448 Bq kg-1 dm). Potential calculated annual effective doses due to mushroom consumption by adults, considering only the effect of 137Cs, reached 0.311 and 8.71 mSv in B. edulis and I. badia from highly contaminated locations, respectively, and 0.0014 and 0.009 mSv in these species from low contaminated ones.

Effect of metal ions on autofluorescence of the dry rot fungus Serpula lacrymans grown on spruce wood.

This work describes autofluorescence of the mycelium of the dry rot fungus Serpula lacrymans grown on spruce wood blocks impregnated with various metals. Live mycelium, as opposed to dead mycelium, exhibited yellow autofluorescence upon blue excitation, blue fluorescence with ultraviolet (UV) excitation, orange-red and light-blue fluorescence with violet excitation, and red fluorescence with green excitation. Distinctive autofluorescence was observed in the fungal cell wall and in granula localized in the cytoplasm. In dead mycelium, the intensity of autofluorescence decreased and the signal was diffused throughout the cytoplasm. Metal treatment affected both the color and intensity of autofluorescence and also the morphology of the mycelium. The strongest yellow signal was observed with blue excitation in Cd-treated samples, in conjunction with increased branching and the formation of mycelial loops and protrusions. For the first time, we describe pink autofluorescence that was observed in Mn-, Zn-, and Cu-treated samples with UV, violet or. blue excitation. The lowest signals were obtained in Cu- and Fe-treated samples. Chitin, an important part of the fungal cell wall exhibited intensive primary fluorescence with UV, violet, blue, and green excitation.

Effects of the soil microbial community on mobile proportions and speciation of mercury (Hg) in contaminated soil.

The precise characterization of the behavior of individual microorganisms in the presence of increased mercury contents in soil is necessary for better elucidation of the fate of mercury in the soil environment. In our investigation, resistant bacterial strains isolated from two mercury contaminated soils, represented by Paenibacillus alginolyticus, Burkholderia glathei, Burkholderia sp., and Pseudomonas sp., were used. Two differently contaminated soils (0.5 and 7 mg kg(-1) total mercury) were chosen. Preliminary soil analysis showed the presence of methylmercury and phenylmercury with the higher soil mercury level. Modified rhizobox experiments were performed to assess the ability of mercury accumulating strains to deplete the mobile and mobilizable mercury portions in the soil by modification; microbial agar cultures were used rather than the plant root zone. A sequential extraction procedure was performed to release the following mercury fractions: water soluble, extracted in acidic conditions, bound to humic substances, elemental, and bound to complexes, HgS and residual. Inductively coupled plasma mass spectrometry (ICP-MS) and a single-purpose atomic absorption spectrometer (AMA-254) were applied for mercury determination in the samples and extracts. Gas chromatography coupled to atomic fluorescence spectrometry (GC-AFS) was used for the determination of organomercury compounds. The analysis of the microbial community at the end of the experiment showed a 42% abundance of Paenibacillus sp. followed by Acetivibrio sp., Brevibacillus sp., Cohnella sp., Lysinibacillus sp., and Clostridium sp. not exceeding 2% abundance. The results suggest importance of Paenibacillus sp. in Hg transformation processes. This genus should be tested for potential bioremediation use in further research.

Translocation of mercury from substrate to fruit bodies of Panellus stipticus, Psilocybe cubensis, Schizophyllum commune and Stropharia rugosoannulata on oat flakes.

The cultivation and fructification of 15 saprotrophic and wood-rotting fungal strains were tested on three various semi-natural medium. The formation of fruit bodies was observed for Panellus stipticus, Psilocybe cubensis, Schizophyllum commune and Stropharia rugosoannulata in the frame of 1-2 months. Mercury translocation from the substrate to the fruit bodies was then followed in oat flakes medium. Translocation was followed for treatments of 0, 1.25, 2.5, 5, 10 and 20ppm Hg in the substrate. All four fungi formed fruit bodies in almost all replicates. The fruit body yield varied from 0.5 to 15.3g dry weight. The highest bioconcentration factor (BCF) of 2.99 was found for P. cubensis at 1.25ppm Hg. The BCF decreased with increasing Hg concentration in the substrate: 2.49, 0, 2.38, 1.71 and 1.82 for P. stipticus; 3.00, 2.78, 2.48, 1.81 and 2.15 for P. cubensis; 2.47, 1.81, 1.78, 1.07 and 0.96 for S. commune; and 1.96, 1.84, 1.21, 1.71 and 0.96 for S. rugosoannulata. The Hg contents in the fruit bodies reflected the Hg contents in the substrate; the highest contents in the fruit bodies were found in P. cubensis (43.08±7.36ppm Hg) and P. stipticus (36.42±3.39ppm).

Concomitant use of polarization and positive phase contrast microscopy for the study of microbial cells.

Polarization and positive phase contrast microscope were concomitantly used in the study of the internal structure of microbial cells. Positive phase contrast allowed us to view even the fine cell structure with a refractive index approaching that of the surrounding environment, e.g., the cytoplasm, and transferred the invisible phase image to a visible amplitude image. With polarization microscopy, crossed polarizing filters together with compensators and a rotary stage showed the birefringence of different cell structures. Material containing algae was collected in ponds in Sýkorice and Zbecno villages (Krivoklát region). The objects were studied in laboratory microscopes LOMO MIN-8 Sankt Petersburg and Polmi A Carl Zeiss Jena fitted with special optics for positive phase contrast, polarizers, analyzers, compensators, rotary stages, and digital SLR camera Nikon D 70 for image capture. Anisotropic granules were found in the cells of flagellates of the order Euglenales, in green algae of the orders Chlorococcales and Chlorellales, and in desmid algae of the order Desmidiales. The cell walls of filamentous algae of the orders Zygnematales and Ulotrichales were found to exhibit significant birefringence; in addition, relatively small amounts of small granules were found in the cytoplasm. A typical shape-related birefringence of the cylindrical walls and the septa between the cells differed in intensity, which was especially apparent when using a Zeiss compensator RI-c during its successive double setting. In conclusion, the anisotropic granules found in the investigated algae mostly showed strong birefringence and varied in number, size, and location of the cells. Representatives of the order Chlorococcales contained the highest number of granules per cell, and the size of these granules was almost double than that of the other monitored microorganisms. Very strong birefringence was exhibited by cell walls of filamentous algae; it differed in the intensity between the cylindrical peripheral wall and the partitions between the cells. Positive phase contrast enabled us to study the morphological relationship of various fine structures in the cell (poorly visible in conventional microscope) to anisotropic structures that have been well defined by polarization microscopy.

Extracellular enzymes of the white-rot fungus Fomes fomentarius and purification of 1,4-ß-glucosidase.

Production of the lignocellulose-degrading enzymes endo-1,4-ß-glucanase, 1,4-ß-glucosidase, cellobiohydrolase, endo-1,4-ß-xylanase, 1,4-ß-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-ß-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml(-1)), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml(-1)). The 1,4-ß-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-ß-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5-5.0, and K (M) and V (max) values were 62 µM and 15.8 µmol min(-1) l(-1), respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K (i) of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0-6.0, respectively. The combination of relatively low molecular mass and low K (M) value make the 1,4-ß-glucosidase a promising enzyme for biotechnological applications.

Ecology of coarse wood decomposition by the saprotrophic fungus Fomes fomentarius.

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high ß-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

Development of soil microbiology methods: from respirometry to molecular approaches.

This review deals with techniques and methods used in the study of the function and development of microorganisms occurring in soil with emphasis on the contributions of Czech Academician Ivan Málek and his coworkers or fellows (Jirí Macura, Frantisek Kunc) to the development of basic techniques used in soil microbiology. Early studies, including batch cultivation and respirometric techniques, as well as later developments of percolation and continuous-flow methods of cultivation of soil microorganisms are discussed. Recent developments in the application of analytical chemistry (HPLC or GC) and of molecular biological techniques to ecological questions that have revolutionized concepts in soil microbiology and microbial ecology are also briefly mentioned, including denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), phospholipid fatty acid analysis (PLFA) and others. The shift of soil microbiology from the study of individual microorganisms to entire microbial communities, including nonculturable species, is briefly discussed.

Degradation of lignocellulose by Pleurotus ostreatus in the presence of copper, manganese, lead and zinc.

Pleurotus ostreatus produces the cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase, endo-1,4-beta-mannanase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase during growth on wheat straw in the presence and absence of Cu, Mn, Pb, and Zn. This is the first report concerning endo-1,4-beta-mannanase in P. ostreatus. Although the concentrations of trace metals in wheat straw ranged from units to tens of microg g(-1), only 3-6% (Fe, Pb) or 30-45% (Cu, Mn, Zn) of the total amount was extractable and available for the fungus. The substrate colonization rate was only decreased by high concentrations of Cu and Zn; the loss of dry mass differed among treatments in the initial phase of fungal growth, and at the end of the experiment (day 98) it was significantly lower in metal-containing treatments (63-66%) than in the control (70%). The cellulolytic and hemicellulolytic enzyme were prone to a metal effect except for the increase in endo-1,4-beta-glucanase and 1,4-beta-glucosidase in the presence of Zn. Laccase activity was increased by all tested metals, and unlike other white-rot fungi, Mn-peroxidase levels were low in the presence of manganese.

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.

ICP-MS determination of heavy metals in submerged cultures of wood-rotting fungi.

Accumulation of five heavy metal ions by five species of wood-rotting basidiomycetes during a 9-day cultivation was studied. Contents of Cd, Cu, Pb, and Zn were measured using ICP-MS; the amount of mercury was determined directly in solid samples using the Advanced Mercury Analyser. A standard operation procedure for the sample preparation and determination of metal content was developed and validated. Presence of Cd, Cu, Hg, and Pb decreased the accumulation of zinc by the fungi. The basidiomycete Pycnoporus cinnabarinus exhibited the highest metal binding capacity of all fungi tested.

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.

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium.

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus.

Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection.

A method based on the coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP MS) was developed for screening the changes in the bioligand composition of wood-rotting fungi as a function of their exposure to copper stress. Strains of four different species of wood-rotting fungi: Phanerochaete chrysosporium, Schizophyllum commune, Daedalea quercina and Pleurotus ostreatus were examined. Only one, namely Ph. chrysosporium, showed any significant difference in terms of the fingerprint of Cu-binding ligands between control and exposed cells which suggest trapping of Cu(II) by cell walls as the only resistance mechanisms. In the case of Ph. chrysosporium the bioinduction of a new Cu-binding ligand was demonstrated. The presence of a new compound in the SE chromatographic fraction of interest was confirmed by CZE-ICP MS. Attempts to identify the new compound by electrospray MS/MS failed because of insufficient sensitivity.

Copper and cadmium increase laccase activity in Pleurotus ostreatus.

Addition of copper (0.5-5 mM) or cadmium (1-5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H(2)O(2) decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05-50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.

Intraspecific variability in growth response to cadmium of the wood-rotting fungus Piptoporus betulinus.

The intraspecific variability in growth response to cadmium (Cd) on agar media and in liquid culture was studied among fourteen strains of a wood-rotting fungus Piptoporus betulinus. The variability of Cd tolerance was found to be very high. The ED(50) ranged from 6.8 µM Cd in the most sensitive strain, up to 255.1 µM in the most resistant one. On agar media the addition of Cd to nutrient media resulted in reduction of relative growth rate and increased lag time. While the reduction of growth rate was already apparent at 10 µM Cd, the lag time was significantly increased in higher Cd concentrations. Five strains of P. betulinus failed to grow at 250 µM Cd and none grew at 500 µM metal. Biomass production in liquid culture was less sensitive to addition of Cd than the growth rate on solid media. At 100 µM Cd the radial growth rate of the mycelium was reduced to 27%, whereas the dry mass of mycelium was 77% of the respective control value. A group of four Cd-sensitive strains was found, showing low metal tolerance both on solid media and in liquid cultures. Although the isolates originated from sites with different Cd-pollution level, no correlation between level of Cd-pollution and resistance (ED(50)) was found. The growth rate of fourteen tested strains displayed lower variability than biomass production, showing that radial growth rate is more species-specific and therefore more valuable for interspecific comparisons of growth response.