Screening of White-Rot Fungi Isolates for Decolorization of Pulp and Paper Mill Effluent and Assessment of Biodegradation and Biosorption Processes.

Ten white-rot fungal isolates were evaluated for the decolorization potential of pulp and paper mill effluent. Trametes elegans PP17-06, Pseudolagarobasidium sp. PP17-33, and Microporus sp.2 PP17-20 showed the highest decolorization efficiencies between 42 and 54% in 5 d. To reveal the mechanisms involved in decolorization and assess the long-term performance, PP17-06, which showed the highest decolorization efficiency, was further investigated. It could reduce the ADMI color scale by 63.6% in 10 d. However, extending the treatment period for more than 10 d did not significantly enhance the decolorization efficiencies. The maximum MnP activity of 3.27 U L-1 was observed on the 6 d during the biodegradation. In comparison, laccase activities were low with the maximum activity of 0.38 U L-1 (24 d). No significant LiP activities were monitored during the experiment. Dead fungal biomass showed an optimum decolorization efficiency of 44.18% in 8 d employing the biosorption mechanism. No significant changes in the decolorization efficiency were observed after that, suggesting the equilibrium status was reached. These results revealed that PP17-06 has the potential to decolorize pulp and paper mill effluent by employing both biodegradation and biosorption processes.

Successful Rescue of Wild Trametes versicolor Strains Using Sawdust and Rice Husk-based Substrate.

<b>Background and Objective:</b> <i>Trametes versicolor</i> has not only been valued in medical use but also in environmental protection. One of the major challenges currently faced in the commercial cultivation of <i>T. versicolor</i> is finding superior strains that can produce high yields. In an attempt to search for high-yield potential <i>T. versicolor</i>, two wild strains, namely VNUA and BV, were isolated and evaluated for potential cultivation. <b>Materials and Methods:</b> Optimized culture conditions were set up by one-individual factor-at-a-time. Four different kinds of culture media, including Czapek, Raper, PGA and modified Potato Dextrose Agar (PDA), were investigated to ascertain the optimal media. The efficiency of sawdust and rice grain for mother spawn production was evaluated. Different combinations of sawdust and rice husk were tested to investigate the most favorable substrate mixtures. <b>Results:</b> The ideal medium and temperature for the favorable mycelial growth of <i>T. versicolor</i> were PGA and 30°C, respectively. The optimal spawning material for upscaling of the mycelium was Treatment D (20% rice grain, 79% sawdust and 1% calcium carbonate). The strains were successfully cultivated in a basal substrate combination of sawdust and rice husk supplemented with wheat bran. Investigated strains responded differently to different substrates cultivation. Of note, compared with strain BV, strain VNUA showed a significantly higher biological efficiency (7.3%). <b>Conclusion:</b> Wild <i>T. versicolor</i> strains were successfully fructified under artificial cultivation conditions. Strain VNUA can be considered as a potential strain for commercial cultivation. The use of sawdust for the spawn production of <i>T. versicolor</i> can reduce the cost of manufacturing.

Notes on Medicinal Polypore Species from the Genus Daedaleopsis (Agaricomycetes), Distributed in the Asian Part of Russia.

We present a study on the medicinal value, taxonomy, and ecology of the polypore mushrooms Daedaleopsis confragosa and D. tricolor isolated from the Asian part of Russia (the Urals, Siberia, and the Far East). The phylogenetic analysis of recombinant DNA internal transcribed spacer sequences data has shown that D. confragosa and D. tricolor do not differ taxonomically and should be considered as one species. However, because D. confragosa and D. tricolor differ significantly in their ecological characteristics, they may be considered as two morpho-ecological varieties: D. confragosa var. confragosa and D. confragosa var. tricolor (both nomen provisiorum).

Bioprocess optimization of laccase production through solid substrate fermentation using Perenniporia tephropora-L168 and its application in bioremediation of triaryl-methane dye.

Laccases are multi copper oxidases that can oxidize both phenolic and nonphenolic lignin related compounds. Consequently, there has been continuous demand for laccases for the oxidative degradation of phenolic dyes in effluents. In view of this, the present work was focused on laccase production by solid substrate fermentation using a newly isolated fungus Perenniporia tephropora-L168. To intensify the laccase production, the process parameters pH, nitrogen, inducer, and substrate: water ratio were optimized by using statistical model. A set of optimal conditions noted were pH 3, nitrogen 0.001 g/L; inducer 0.5% and substrate: water ratio (1:10), which yielded laccase 1,160 U/g. The crude laccase exhibited noteworthy potential to degrade a triaryl-methane dye especially Malachite green. Also, during bioremediation studies, the statistical process optimization could achieve 81% decolourization within 180 min. The laccase treatment brought chemical transformation in malachite green as evident from UV-Visible spectra, FTIR, HPLC while toxicity against bacteria and fungi was also reduced. During phytotoxicity study, effect of treated and untreated dye on germination of seed was analyzed. Interestingly, the germination index for Vigna aconitifolia and Vigna radiata was increased by two and fourfold, respectively. Overall, this work demonstrates optimized production of laccase using Perenniporia tephropora-L168 and its efficient bioremediation potential for triaryl-methane dye.

Solid state fermentation and crude cellulase based bioconversion of potential bamboo biomass to reducing sugar for bioenergy production.

BACKGROUND: Lignocellulosic biomass from bamboo is an attractive feedstock for the bioethanol industry owing to its high cellulosic content and fast growth rate. In this study, powdery biomass was first enzymatically delignified and then saccharified using crude enzymes. RESULTS: The biological pretreatment decreased the lignin content of the biomass from an initial value of 295 to 137.7 g kg-1 , with a simultaneous increase in exposed cellulose content from 379.3 to 615.9 g kg-1 . For optimization of the saccharification, response surface methodology was adopted using a three-factor/three-level Box-Behnken design with crude fungal cellulase loading (FPU g-1 substrate), substrate concentration (% w/v) and saccharification temperature (°C) as the main process parameters. A maximum saccharification yield of 47.19% was achieved under the optimized conditions (cellulase enzyme 18.4 FPU g-1 substrate, substrate concentration 1.0% w/v, temperature 39.49 °C). Biological delignification and saccharification of the biomass were further confirmed through scanning electron microscopy analysis. CONCLUSION: It is evident from the study that bamboo, as a renewable energy bioresource, can be hydrolysed to reducing sugars by using crude laccase/cellulase enzymes of fungal origin with good saccharification yield. Thus crude enzyme preparations could be utilized efficiently for eco-friendly and cost-effective bioethanol production. © 2018 Society of Chemical Industry.

Purification and characterization of a novel extracellular neutral metalloprotease from Cerrena albocinnamomea.

We selected a fungus secreting a neutral protease from soil and identified it as the basidiomycete fungus Cerrena albocinnamomea according to its ITS-5.8S rDNA and 28S rDNA-D1/D2 sequences. A major extracellular protease isolated from C. albocinnamomea was purified approximately 44-fold through two purification steps. SDS-PAGE analyses of the purified protease revealed a single band, and its molecular mass of 39,756 Da was determined using MALDI-TOF-MS. The enzyme was optimally active at approximately pH 7.0 and 45°C. The Km and Vmax values for the hydrolysis of azocasein were 2.46 mg/mL and 989 units/min/mg protein, respectively. The enzyme was stable at pH 3.6-8.6 for 16 h and at temperatures ≤35°C for 1 h. Enzymatic activity was completely inhibited by Cu2+ and Zn2+ and markedly by EDTA and phosphoramidon. The N-terminal amino acid sequence ASYRVLPIT is highly similar to those of the members of the metalloprotease family M36, such as keratinase and elastinase. However, the protease did not detectably hydrolyze keratin or elastin. In contrast, the protease hydrolyzed fibrinogen, although there were no significant sequence similarities to the N-terminal amino acid sequences of other fibrinolytic enzymes. These results suggest that the purified protease represents a new neutral metalloprotease with fibrinogenolytic activity.

The molecular response of the white-rot fungus Dichomitus squalens to wood and non-woody biomass as examined by transcriptome and exoproteome analyses.

The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post-)genomic analyses have shown that species that occupy a large variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well-adapted to the prevailing substrate. White-rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white-rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non-woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non-woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat.

Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production.

Molecular phylogeny and morphology reveal a new species of Amyloporia (Basidiomycota) from China.

Amyloporia pinea sp. nov. is described and illustrated on the basis of collections from southern China. Morphology and phylogenetic analysis of rDNA ITS sequences support this new species. Morphologically, it is characterized by resupinate, annual basidiocarps, cream to yellowish buff pore surface when fresh, which becomes yellowish brown to clay-buff upon drying, a dimitic hyphal system with clamped generative hyphae and inamyloid skeletal hyphae, fusoid cystidioles, and cylindrical basidiospores; moreover, it causes a brown rot. Molecular phylogeny inferred from ITS sequence data suggested a close relationship between A. pinea and Amyloporia crassa sensu lato. Antrodia subxantha has amyloid skeletal hyphae, and grouped within the Amyloporia clade, hence, it is transferred to Amyloporia, and a new combination Amyloporia subxantha is proposed.

Cytotoxic metabolites from Perenniporia tephropora, an endophytic fungus from Taxus chinensis var. mairei.

Based on bioactivity-oriented isolation, the EtOAc extract of a culture broth of the endophytic fungus Perenniporia tephropora Z41 from Taxus chinensis var. mairei, with strong anti-Pyricularia oryzae activity, afforded a new sesquiterpenoid, perenniporin A (1), together with three known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]genfuran-5-ol (3), and albicanol (4). Their structures were elucidated by means of spectroscopic methods. All the isolated compounds and the EtOAc extract of P. tephropora Z41 (EPT) were evaluated for their cytotoxic activity against three human cancer cell lines (HeLa, SMMC-7721, and PANC-1). EPT demonstrated significant cytotoxicity with IC(50) values ranging from 2 to 15 µg/mL. Compound 2 was the most cytotoxic constituent against the tested cell lines with IC(50) values of 1.16, 11.63, and 11.80 µg/mL, respectively, while compounds 1, 3, and 4 exhibited moderate cytotoxicity with IC(50) values ranging from 6 to 58 µg/mL. We conclude that the endophytic fungus P. tephropora is a promising source of novel and cytotoxic metabolites.

First human case of pulmonary fungal ball due to a Perenniporia species (a basidiomycete).

Perenniporia species are basidiomycetes, resupinate shelf fungi responsible for white rot decay of wood. Here, we report for the first time an intracavitary pulmonary fungal ball due to a species of Perenniporia that has not been recognized so far as a human pathogen. The fungus was identified by sequencing of the partial ribosomal operon of a culture from a clinical specimen.

Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat.

A thermotolerant and halotolerant strain of Pycnoporus sanguineus was isolated from an oil-polluted site in a tropical area located in Veracruz, Mexico. This strain was able to grow at 47 degrees C and in culture medium containing 500 mM NaCl. The strain was also tolerant to the presence of 30,000 ppm of crude Maya oil. A 68-kDa protein purified from submerged cultures exhibited laccase activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine, and o-dianisidine, for which it presented the highest affinity (Km = 43 microM). Two-dimensional gel electrophoresis analysis showed that, unusual for laccases, the enzyme has two active isoforms, with isoelectric points of 7.00 and 7.08. The purified enzyme showed high thermostability, retaining 40% of its original activity after 3 h at 60 degrees C. This property seems to correlate with a long "shelf-life," given that at 40 degrees C enzyme activity was only gradually lost over a 5-day period incubation. Both the fungus and its laccase are likely to have high potential for biotechnological applications.

Phylogenetic relationships of Polyporus and morphologically allied genera.

Polyporus accommodates morphologically heterogeneous species and is divided into six infrageneric groups based on macromorphological characters. On the other hand allied genera have macro- and microscopic characters similar to those of Polyporus. The phylogenetic relationships of Polyporus and allied genera were established from sequences of RNA polymerase II second largest subunit (RPB2), nuclear ribosomal large subunit (nucLSU) and mitochondrial ATPase subunit 6 (ATP6). The molecular phylogenetic trees confirmed that Polyporus is a polyphyletic genus and recognized six major clades (1-6) containing species of Polyporus and several allied genera. Among the clades one contained three infrageneric groups of Polyporus and two allied genera, Datronia and Pseudofavolus while one other contained group Polyporellus and Lentinus. Five of the six major clades contained species belonging to a single infrageneric group, Favolus, Melanopus, Polyporellus or Polyporus. This suggests that morphological characters used to define these groups have phylogenetic significance and reveals the need for a taxonomic revision of Polyporus and its allied genera.

Species diversity of polyporoid and corticioid fungi in northern hardwood forests with differing management histories.

Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.

New lanostane-type triterpenes from Fomes officinalis.

Five new lanostane-type triterpenes, named fomefficinic acid A-E (1-5), were isolated from the dried sclerotium of Fomes officinalis, respectively. Their structures were established as 24-methylene-3-oxo-lanost-8-en-21-oic acid (1), 3alpha,15alpha-dihydroxy-24-methylene-lanosta-7,9(11)-dien-21-oic acid (2), 3alpha,15alpha-dihydroxy-24-methylene-lanost-8-en-21-oic acid (3), 15alpha-hydroxy-3-oxo-24-methylenelanost-8-en-21-oic acid (4), 15alpha-acetoxy-3-oxo-24-methylenelanosta-7,9(11)-dien-21-oic acid (5), by spectral analysis and chemical methods as well as comparison with known compounds.

Screening for pectinolytic activity of wood-rotting basidiomycetes and characterization of the enzymes.

Seventy-five fungal strains from different groups of basidiomycetes, newly isolated from rotten wood, were screened for pectinolytic activity. Despite the fact that basidiomycetes are scarcely referred to as pectinase producers, the polygalacturonase (PG) activity was detected in 76% of the strains; 16% with activity higher than 40 nkat/g, 40% between 13.3 and 40 nkat/g, and 44% with activity lower than 13.3 nkat/g. The highest productions were obtained among the fungi from order Aphyllophorales, family Polyporaceae. The characterization of the enzymes from the highest PG producers (Lentinus sp., Gloeophyllum striatum, Pycnoporus sanguineus, Schizophyllum commune) showed optimum temperature for catalytic activity at 60-70 degrees C and two peaks of pH optimum (3.5-4.5 and 8.5-9.5). The enzymes exhibited high pH stability (3.0-11.0) but after incubation at 40 degrees C for 1 h their activity dropped by 18-73%.

Genetic differentiation in Fomitopsis pinicola (Schwarts: Fr.) Karst studied by means of arbitrary primed-PCR.

Genetic variation within and among one Finnish and three Swedish populations of Fomitopsis pinicola (Schwarts: Fr.) Karst. were studied by amplifying DNA from haploid isolates originating from single spore cultures using two arbitrary primers. Analysis offspring from single fruit bodies revealed only three pairs of codominant alleles among 42 variable genetic markers, the remaining 38 segregated independently. Genetic similarity was measured in terms of Euclidean distance. Individuals in the Finnish population tended to form a distinct cluster in the principal component analysis. Variation within and among populations/regions was partitioned by Analysis of Molecular Variance-AMOVA. Within population variation accounted for 91.6% of the total genetic variation. The remaining 7.68% was accounted for by variation between the Finnish population and each of the three Swedish ones. Variation among the Swedish populations accounted for only 0.72% of the total variation. Wright's Fst was 0.17 for all four populations and 0.13 for the three Swedish populations. These relatively low values indicate that there is gene flow among all populations or that they are derived from a common ancestral population. The observed pattern of genetic variation is probably the result of effective spore dispersal and the continuous distribution of this common early successional species.

Growth and production phases of pycnoporus sanguineus

Foi observado que na cultura de Pycnoporus sanguineus MIP 89007, a síntese de substâncias com atividade antimicrobiana ocorreu principalmente entre o 18§. e o 23§. dia de incubaçäo. Além disso, foi também constatado que a substância produzida foi rapidamente degradada quando permaneceu no caldo de cultura após ter cessado a síntese e que os extratos obtidos a partir do fungo somente retiveram a atividade quando mantidos à vácuo

Antitumor activity of a polysaccharide fraction extracted from cultured fruiting bodies of Grifola frondosa.

Antitumor activity of a polysaccharide fraction (GF-1) extracted from cultured fruiting bodies of a fungus, Grifola frondosa, was examined on allogeneic and syngeneic tumors in mice. GF-1 had a marked inhibitory activity against the growth of subcutaneously (s.c.) inoculated Sarcoma 180 by the intraperitoneally (i.p.) injection at 0.5-5.0 mg/mouse for 10 successive days. A significant antitumor activity was also observed when GF-1 at 4.0 mg/mouse was i.p. injected successively on days +1-+5, +7-+11, +14-+18 or +21-+25 if the tumor cells were inoculated s.c. on day 0. Similar results were obtained by a single i.p. injection of GF-1 at 2.0 mg/mouse on day +1, +7, +14 or +21. When GF-1 was injected i.p., intravenously (i.v.) or intratumorally (i.t.), all of them showed an equivalent level of higher inhibitory activity (inhibition ratio; over 90%). However, the oral (p.o.) administration was not effective. The pretreatment of mice with GF-1 at 2.0 or 4.0 mg/mouse for 5 times before tumor inoculation did not show a significant antitumor activity. In addition, GF-1 administered i.p. at varying times before and/or after the tumor inoculation showed no antitumor activity against ascites form of Sarcoma 180. In the syngeneic systems, GF-1 exhibited an antitumor activity against solid form of Meth A fibrosarcoma in BALB/c mice and MM46 carcinoma in C3H/He mice.