Mycoremediation of oil contaminant by Pleurotus florida (P.Kumm) in liquid culture.

This study investigated the potential functions of Pleurotus florida (an edible mushroom) in the biodegradation of gas oil at concentrations of 0 (control), 2.5, 5, and 10% (V: V) for 30 days. The gas oil increased dry weight and protein concentration in all treatments (by an average of 19.5 and 108%, respectively). Moreover, the pH, surface tension (ST), and interfacial tension (IFT) were reduced by the mushroom supplementation. The lowest surface tension (31.9 mN m-1) and the highest biosurfactant production belonged to the 10% gas oil treatment (0.845 ± 0.03 mg mL-1). The results demonstrated that the adsorption isotherm agreed well with the Langmuir isotherm. The maximum Langmuir adsorption capacity was calculated at 0.743 mg g-1 wet biomass of P. florida. The fungal supplementation efficiently remedied the total petroleum hydrocarbons (TPHs) by an average of 55% after 30 days. Gas chromatography (GC) analysis revealed that P. florida effectively detoxified C13-C28 hydrocarbons, Pristane, and Phytane, implying its high mycoremediation function. The toxicity test showed that mycoremediation increased the germination by an average of 35.82% ± 8.89 after 30 days. Laccase activity increased significantly with increasing gas oil concentration in the treatments. The maximum laccase activity was obtained in the 10% gas oil treatment (142.25 ± 0.72 U L-1). The presence of pollutants was also associated with induction in the tyrosinase activity when compared to the control. These results underline the high mycoremediation capacity of P. florida through the involvement of biosurfactants, laccase, and tyrosinase.

Mycelial Growth and Antimicrobial Activity of Pleurotus Species (Agaricomycetes).

The objective of this study was to analyze mycelial growth under different culture conditions and antimicrobial activity of Pleurotus ostreatus (Jacq.: Fr.) Kumm. (DPUA 1533) and P. ostreatus (Jacq.: Fr.) Kumm. cv. Florida (DPUA 1534) against fungi and bacteria of medical importance. The growth of Pleurotus species was evaluated in natural and complex media, with and without light, at 25°C and 28°C for 8, 15, and 30 days. Candida albicans (DPUA 1336), Cryptococcus laurentii (DPUA 1501), Aspergillus flavus (DPUA 1836), Escherichia coli (DAUPE 224), and Mycobacterium smegmatis (ATCC 607) were used to test antibiosis. Under all growth conditions in vitro, Pleurotus species evidenced growth and high density of mycelia on potato dextrose agar and Sabouraud agar with yeast extract; mycelial growth but lesser mycelial density was observed on rice bran extract agar. Organic mycelial extracts of Pleurotus species exhibited potential antibacterial and antifungal activity, and were selective for the tested microorganisms.

Benzoic acid derivatives with improved antifungal activity: Design, synthesis, structure-activity relationship (SAR) and CYP53 docking studies.

Previously, we identified CYP53 as a fungal-specific target of natural phenolic antifungal compounds and discovered several inhibitors with antifungal properties. In this study, we performed similarity-based virtual screening and synthesis to obtain benzoic acid-derived compounds and assessed their antifungal activity against Cochliobolus lunatus, Aspergillus niger and Pleurotus ostreatus. In addition, we generated structural models of CYP53 enzyme and used them in docking trials with 40 selected compounds. Finally, we explored CYP53-ligand interactions and identified structural elements conferring increased antifungal activity to facilitate the development of potential new antifungal agents that specifically target CYP53 enzymes of animal and plant pathogenic fungi.

Potential of Pleurotus ostreatus mycelium for selenium absorption.

The aim of this study was to evaluate the effect of high selenium (Se) concentrations on morphophysiological and ultrastructural properties of Pleurotus ostreatus. Mycelium growth was good in media enriched with 5.0, 10.0, and 20.0 mg L(-1) of Se, concentration of 500.0 mg L(-1) strongly inhibited growth, and 1000.0 mg L(-1) was the minimum inhibitory concentration. Contrary to thin-walled, hyaline, branched, and anastomized hyphae with clamp-connections in the control, at Se concentrations of 100.0 and 500.0 mg L(-1), they were noticeably short, frequently septed and branched, with a more intensive extracellular matrix, and without clamp-connections. At high Se concentrations, hyphae with intact membrane, without cellular contents, with a high level of vacuolization, and with numerous proteinaceous bodies were observed. Biomass yield ranged between 11.8 g L(-1), in the control, and 6.8 g L(-1), at an Se concentration of 100.0 mg L(-1), while no production was detected at a concentration of 500.0 mg L(-1). Se content in the mycelia reached a peak (938.9 µg g(-1)) after cultivation in the medium enriched with Se at the concentration of 20.0 mg L(-1), while the highest absorption level (53.25%) was found in the medium enriched with 5.0 mg L(-1) Se.

Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii.

This study was conducted to evaluate the physiological effects of different selenium (Se) levels on the growth of white-rot fungus, Pleurotus eryngii, with special reference to the regulation of ligninolytic enzymes such as laccase and versatile peroxidase. The fungus was grown in medium supplemented with 1, 10, 100, 1,000 and 10,000 µM of sodium selenite. Mycelial growth was stronger at lower Se levels, but declined significantly at higher concentrations of 1,000 and 10,000 µM, highlighting its association in mediating toxic responses. Inhibition of fungal growth was accompanied with dense and entangled hyphae taking the shape of irregular short strips. Additionally, hyphal swellings and septation were noticed which lead to a reduction in the advancement of the mycelium. Along with the inhibition of fungal biomass, the reducing sugar and protein concentrations increased to about 30.2 and 3.5 mg/ml respectively in the growth medium. Additionally, the laccase gene expression showed a twofold upregulation at higher levels of Se, although the activity of the enzyme was compromised with an inverse relationship with increased gene transcripts. The versatile peroxidase transcript showed a complete downregulation at 10,000 µM after an upregulation at lower levels of Se. We also confirmed the direct relationship of different Se levels on laccase activity of Rhus vernicifera that showed similar behavior to the fungal laccase. The results of the present study suggest that Se supplementation regulates mRNA levels of laccase and versatile peroxidase depending on exposure and may play a role in the toxicity associated with Se.

Pleurotus ostreatus biofilms exhibit higher tolerance to toxicants than free-floating counterparts.

The MBEC(TM)-High Throughput Assay based on the Calgary Biofilm Device was used to produce and to characterize Pleurotus ostreatus biofilms. Hydroxyapatite coating of pegs was required to enable biofilm attachment; biofilm amounts and homogeneity of distribution were markedly improved upon removal of non-sessile biomass after 48 h from inoculation. Scanning electron microscopy showed surface-associated and multi-layered growth stabilized by the presence of an extracellular matrix (ECM). Biofilms had higher contents of total sugars and ECM than their free-floating counterparts. Tolerance to Cr(VI) in the former was about twice that of the latter as inferred by the respective inhibitory concentrations (48.4 vs 24.1 mM and 114.5 vs 61.0 mM in 4- and 7-d-old cultures, respectively). Biofilms also displayed superior olive-mill wastewater (OMW) treatment efficiency along 5 consecutive batches leading to chemical oxygen demand and total phenol removals higher than 50 and 90%, respectively. Laccase activity peaks in biofilm cultures grown on OMW were significantly higher than those in free-floating cultures.

Heat treatment of wheat straw by immersion in hot water decreases mushroom yield in Pleurotus ostreatus.

BACKGROUND: The oyster mushroom, Pleurotus ostreatus, is cultivated worldwide. It is one of the most appreciated mushrooms due to its high nutritional value. Immersion of the substrate in hot water is one of the most popular and worldwide treatment used for mushroom farmers. It is cheap and easy to implement. AIMS: To compare the yields obtained during mushroom production of P. ostreatus using different pre-treatments (immersion in hot water, sterilization by steam and the use of fungicide) to determine if they influence mushroom crop. METHODS: Four different treatments of substrate (wheat straw) were carried out: (i) immersion in hot water (IHW); (ii) steam sterilization; (iii) chemical; and (iv) untreated. The residual water from the IHW treatment was used to evaluate the mycelium growth and the production of P. ostreatus. RESULTS: Carbendazim treatment produced highest yields (BE: 106.93%) while IHW produced the lowest BE with 75.83%. Sugars, N, P, K and Ca were found in residual water of IHW treatment. The residual water increased the mycelium growth but did not increase yields. CONCLUSIONS: We have proved that IHW treatment of substrate reduced yields at least 20% when compared with other straw treatments such as steam, chemical or untreated wheat straw. Nutrients like sugars, proteins and minerals were found in the residual water extract which is the resultant water where the immersion treatment is carried out. The loss of these nutrients would be the cause of yield decrease. Alternative methods to the use of IHW as treatment of the substrate should be considered to reduce economical loss.

Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn(2+) supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn(2+) on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn(2+)-deficient media, whereas strongly repressed (to approximately 1%) in Mn(2+)-supplemented media. Accordingly, in-vitro Mn(2+)-independent activity was found to be negligible. We tested whether release of mnp4 from Mn(2+) repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the ß-tubulin promoter was produced. Now, despite the presence of Mn(2+) in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to ß-tubulin) and the activity was comparable to the typical activity of PC9 in Mn(2+)-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [(14)C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn(2+) suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

Influence of calcium and silicon supplementation into Pleurotus ostreatus substrates on quality of fresh and canned mushrooms.

Supplements of gypsum (calcium source), pumice (silicon source) and pumice sulfate (silicon and calcium source) into substrates for oyster mushrooms (Pleurotus ostreatus) were searched for their effects on production as well as qualities of fresh and canned mushrooms. The addition of pumice up to 30% had no effect on total yield, size distribution and cap diameters. The supplementation of gypsum at 10% decreased the total yield; and although gypsum at 5% did not affect total yield, the treatment increased the proportion of large-sized caps. High content (>10%) of pumice sulfate resulted in the lower yield. Calcium and silicon contents in the fruit bodies were not influenced by supplementations. The centrifugal drip loss values and solid content of fresh mushrooms, and the percentage of weight gained and firmness of canned mushrooms, cultivated in substrates supplemented with gypsum, pumice and pumice sulfate were significantly (p≤0.05) higher than those of the control. Scanning electron micrographs revealed the more compacted hyphae of mushroom stalks supplemented with silicon and/or calcium after heat treatment, compared to the control. Supplementation of P. ostreatus substrates with 20% pumice was the most practical treatment because it showed no effect on yield and the most cost-effective.

Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 26⁻² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.

Effect of asafoetida extract on growth and quality of Pleurotus ferulic.

Different concentrations of asafoetida extract were added to the medium of Pleurotus ferulic and the effects of the extract on growth of P. ferulic mycelium and fruiting bodies was observed. As the amount of asafoetida extract additive was increased, the growth of Pleurotus mycelium was faster, the time formation of buds was shorter and that yield of fruiting bodies was stimulated. However, overdosing of asafoetida extract hampered the growth of Pleurotus ferulic. The amino acid composition and volatile components in three kinds of pleurotus' were contrasted, including wild pleurotus (WP), cultivated pleurotus with asafoetida extract (CPAE) and cultivated pleurotus without asafoetida extract (CP). CPAE with 2.3 g/100 g asafoetida extract addition had the highest content of total amino acids, as well as essential amino acids. WP had a higher content of total amino acids and essential amino acids than CP. In addition, CPAE with 2.3 g/100 g had the highest score of protein content of pleurotus fruiting bodies, while WP had a higher score than CP. In the score of essential amino acid components of pleurotus fruiting bodies, CP had the highest score, while CPAE was higher than WP. Asafoetida extract influenced the volatile components of Pleurotus ferulic greatly, making the volatile components of cultivated pleurotus more similar to those of wild pleurotus (WP).

The protective effect of selenium inorganic forms against cadmium and silver toxicity in mycelia of Pleurotus ostreatus.

The effect of two inorganic selenium forms has been investigated in the mycelia of Pleurotus ostreatus exposed to cadmium and silver salts in the shaken cultures. The degree of toxicity was assessed by the determination of malondialdehyde (MDA; a common biomarker of lipid peroxidation). The mycelia were exposed to one element form (up to 5 mg l(-1)) and also to the following combinations: cadmium(II) + selenium(IV); cadmium(II) + selenium(VI); silver(I) + selenium(IV); silver(I) + selenium(VI). The concentrations of cadmium, silver, selenium, and MDA were assessed in the mixed cytosol and cell membrane fractions (CCM). A positive correlation between MDA and cadmium was found in the CCM (beta=0.7775, P=0.0001), whereas the effect of silver was less significant (beta=0.4642, P=0.039). These results indicate that silver(I) and cadmium(II) have different capacities to induce lipid peroxidation in P. ostreatus. The protective role of selenium against metal-induced oxidative damage was found to be dependent on the oxidation state of the element form in the growth medium. The strongest beneficial effect was observed in mycelia exposed to cadmium(II) + selenium(IV) (inverse correlation between MDA and selenium in the CCM: beta=-0.7129, P=0.009) and it has been ascribed to a lower incorporation of the toxic metal and/or to possible intracellular interaction between selenium and cadmium. Under exposure to silver(I), the protective effect of selenium(IV) was less noticeable (correlation between MDA and selenium in the CCM; beta=-0.6068, P=0.036); in the presence of selenium(VI), no beneficial effect was observed.

Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus.

Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that poxa1b mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between the poxa1b transcript and protein amounts can be explained by the presence of a high level of the protein in P. ostreatus cellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that its physiological activity could occur inside the cell or on the cell wall. Moreover, the POXA1b isoenzyme behaved uniquely, as its activity was maximal on the second day of growth and then decreased. An analysis performed with protease inhibitors revealed that the loss of extracellular POXA1b activity could have been due to the presence of specific proteases secreted into the copper-containing culture medium that affected the extracellular POXA1b isoenzyme.

First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity.

A protein, with a molecular weight of 20 kDa, and an N-terminal sequence analogous to those of thaumatin-like proteins (TLPs) and thaumatins, was first isolated from the legume of the French bean Phaseolus vulgaris cv Kentucky wonder using a simple procedure involving affinity and ion exchange chromatography. The protein was adsorbed on both CM-Sepharose and Affi-gel Blue Gel. It was the first leguminous TLP-like protein demonstrated to exert antifungal activity against Fusarium oxysporum, Pleurotus ostreatus, and Coprinus comatus but not against Rhizoctonia solani.