Biogenic Silver Nanoparticles Associated With Silver Chloride Nanoparticles (ag@agcl) Produced By Laccase From Trametes Versicolor.

In the present study, semi-purified laccase from Trametes versicolor was applied for the synthesis of silver nanoparticles, and the properties of the produced nanoparticles were characterized. All of the analyses of the spectra indicated silver nanoparticle formation. A complete characterization of the silver nanoparticles showed that a complex of silver nanoparticles and silver ions was produced, with the majority of the particles having a Ag(2+) chemical structure. A hypothetical mechanistic scheme was proposed, suggesting that the main pathway that was used was the interaction of silver ions with the T1 site of laccase, producing silver nanoparticles with the concomitant inactivation of laccase activity and posterior complexing with silver ions.

Avaluació del creixement del fong ligninolític Trametes versicolor sobre diferents suports sòlids per a l'aplicació en tractaments de bioremediació

Els fongs de podridura blanca (WRF - de l'anglès: White-rot fungi) són de gran interès en l'àmbit de la bioremediació per la seva capacitat de degradar la lignina. La lacasa, un dels enzims extracel·lulars que aquests fongs excreten per degradar la lignina, pot ser utilitzada per degradar els contamiants presents en una matriu donada. Trametes versicolor ha estat estudiat per la seva capacitat de produïr aquest enzim en rersidus agrícoles com a substrats. Un primer triatge basat en la producció de CO2, la mesura de l'activitat lacasa i la quantificació de l'ergosterol han permés seleccionar els substrats on es donava un major poder oxidatiu dels cultius inoculats amb T. versicolor. Els posteriors experiments de colonització de sòls, on es monitoritzava l'activitat lacasa i l'ergosterol, han mostrat que T. vesicolor és capaç de colonitzar sòls i que té una major activitat lacasa en condicions no estèrils. També h'ha provat, mitjançant el test ND24, que T. versicolor és capaç de degradar un contaminant emergent, el naproxè, en sòls estèrils i no estèrils esmenats amb residus agrícoles.

Non conventional biological treatment based on Trametes versicolor for the elimination of recalcitrant anticancer drugs in hospital wastewater

This work presents a study about the elimination of anticancer drugs, a group of pollutants considered recalcitrant during conventional activated sludge wastewater treatment, using a biological treatment based on the fungus Trametes versicolor. A 10-L fluidized bed bioreactor inoculated with this fungus was set up in order to evaluate the removal of 10 selected anticancer drugs in real hospital wastewater. Almost all the tested anticancer drugs were completely removed from the wastewater at the end of the batch experiment (8 d) with the exception of Ifosfamide and Tamoxifen. These two recalcitrant compounds, together with Cyclophosphamide, were selected for further studies to test their degradability by T. versicolor under optimal growth conditions. Cyclophosphamide and Ifosfamide were inalterable during batch experiments both at high and low concentration, whereas Tamoxifen exhibited a decrease in its concentration along the treatment. Two positional isomers of a hydroxylated form of Tamoxifen were identified during this experiment using a high resolution mass spectrometry based on ultra-high performance chromatography coupled to an Orbitrap detector (LTQ-Velos Orbitrap). Finally the identified transformation products of Tamoxifen were monitored in the bioreactor run with real hospital wastewater

Protein-Polysaccharides of trametes versocolor: production and biological activities

Extracellular-(E-PPS) and intracellular-protein-polysaccharides (I-PPS) complexes were produced by Trametes versicolor in submerged cultures with different carbon sources. The highest extracellular-(EPS) and intracellular-polysaccharide (IPS) concentration in the complexes was obtained with tomato pomace culture. DPPH radical scavenging for E-PPS and I-PPS produced by liter of culture was equivallent to 2.115 +/- A 0.227 and 1.374 +/- A 0.364 g of ascorbic acid, respectively. These complexes showed a protector effect in the oxidation of erythrocyte membranes and had ability to inhibit the hemolysis and methemoglobin synthesis in stressed erythrocytes. These results suggest that extracellular- and intracellular- polysaccharides produced are important bioactive compounds with medicinal potential.

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe(3+)-reductants. Phenolates were the major compounds with Fe(3+)-reducing activity in both fungi and displayed Fe(3+)-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe(3+) and H(2)O(2) (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum-a model brown rot fungus-other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.

Material properties and nondestructive evaluation of laminated veneer lumber (LVL) made from Pinus oocarpa and P. kesiya

This study aimed at evaluating the mechanical, physical and biological properties of laminated veneer lumber (LVL) made from Pinus oocarpa Schiede ex Schltdl (PO) and Pinus kesiya Royle ex Gordon (PK) and at providing a nondestructive characterization thereof. Four PO and four PK LVL boards from 22 randomly selected 2-mm thickness veneers were produced according to the following characteristics: phenol-formaldehyde (190 g/m(2)), hot-pressing at 150A degrees C for 45 min and 2.8 N/mm(2) of specific pressure. After board production, nondestructive evaluation was conducted, and stress wave velocity (v (0)) and dynamic modulus of elasticity (E (Md) ) were determined. The following mechanical and physical properties were then evaluated: static bending modulus of elasticity (E (M) ), modulus of rupture (f (M) ), compression strength parallel to grain (f (c,0)), shear strength parallel to glue-line (f (v,0)), shear strength perpendicular to glue-line (f (v,90)), thickness swelling (TS), water absorption (WA), and permanent thickness swelling (PTS) for 2, 24, and 96-hour of water immersion. Biological property was also evaluated by measuring the weight loss by Trametes versicolor (Linnaeus ex Fries) Pilat (white-rot) and Gloeophyllum trabeum (Persoon ex Fries.) Murrill (brown-rot). After hot-pressing, no bubbles, delamination nor warping were observed for both species. In general, PK boards presented higher mechanical properties: E (M) , E (Md) , f (M) , f (c,0) whereas PO boards were dimensionally more stable, with lower values of WA, TS and PTS in the 2, 24, and 96-hour immersion periods. Board density, f (v,0), f (v,90) and rot weight loss were statistically equal for PO and PK LVL. The prediction of flexural properties of consolidated LVL by the nondestructive method used was not very efficient, and the fitted models presented lower predictability.

Laccase–Prussian blue film–graphene doped carbon paste modified electrode for carbamate pesticides quantification

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussianblue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectro- scopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device.The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 23 full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0,LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra-and inter-day repeatability (1.8–3.8% RSD), reproducibility (4.1 and 6.3%RSD),selectivity(13.2% bias at the higher interference: substrate ratios tested),accuracy and stability(ca. twenty days)for quantification of five carbamates widely applied on tomato and potato crops.The attained detection limits ranged between 5.2×10−9 mol L−1(0.002 mg kg−1 w/w for ziram)and 1.0×10−7 mol L−1 (0.022 mg kg−1 w/w for carbofuran).Recovery values for the two tested spiking levels ranged from 90.2±0.1%(carbofuran)to 101.1±0.3% (ziram) for tomato and from 91.0±0.1%(formetanate)to 100.8±0.1%(ziram)for potato samples.The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Towards the rational biosynthesis of substituted phenazines and phenoxazinones by laccases

Laccases are multi-copper oxidases that oxidise a wide range of substrates including phenol and aniline derivatives, which could be further involved in coupling reactions leading to the formation of dimeric and trimeric structures. This paper describes the enzyme-mediated dimerisation of several ortho and meta, para-disubstituted aromatic amines into phenazine ("head-to-tail" dimers) and phenoxazinone chromophores. The redox properties of substituted aromatic amines were studied by cyclic voltammetry and the kinetic constants of CotA and Trametes versicolor laccases were measured for selected aromatic amines. The structure of novel enzymatically synthesised phenazine and phenoxazinone dyes using CotA laccase was assessed by NMR and MS. Overall our data show that this enzymatic green process is an efficient alternative to the classic chemical oxidation of aromatic amines and phenols, with an impact on the broad field of applications of these heterocyclic compounds.

Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccases

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and H-1 NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzyme's properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed.

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

Experimental calcium oxalate crystal production and dissolution by selected wood rot fungi.

Twenty-six species of white-rotting Agaricomycotina fungi (Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca2+, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species (Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening (Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species (P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 μg mm−2 (at the maximum accumulation rate) to 0.30 μg mm−2. The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species (Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca2+ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.

Treatment of micropollutants in municipal wastewater: study of a sequential batch biofilter

All the experimental part of this final project was done at Laboratoire de Biotechnologie Environnementale (LBE) from the École Polytechnique Fédérale de Lausanne (EPFL), Switzerland, during 6 months (November 2013- May 2014). A fungal biofilter composed of woodchips was designed in order to remove micropollutants from the effluents of waste water treatment plants. Two fungi were tested: Pleurotus ostreatus and Trametes versicolor in order to evaluate their efficiency for the removal of two micropollutants: the anti-inflammatory drug naproxen and the antibiotic sulfamethoxazole,. Although Trametes versicolor was able to degrade quickly naproxen, this fungus was not any more active after one week of operation in the filter. Pleurotus ostreatus was, on contrary, able to survive more than 3 months in the filter, showing good removal efficiencies of naproxen and sulfamethoxazole during all this period, in tap water but also in real treated municipal wastewater. Several other experiments have provided insight on the removal mechanisms of these micropollutants in the fungal biofilter (degradation and adsorption) and also allowed to model the removal trend. Fungal treatment with Pleurotus ostreatus grown on wood substrates appeared to be a promising solution to improve micropollutants removal in wastewater.

Isolamento e seleção de fungos causadores da podridão-branca da madeira em florestas de Eucalyptus spp. com potencial de degradação de cepas e raízes

Este trabalho objetivou isolar fungos causadores da podridão-branca da madeira, a partir de basidiocarpos e de fragmentos de madeira de eucalipto coletados em várias regiões do país, bem como testar seu potencial de degradação de cepas e raízes mortas em plantios comerciais de eucalipto, após o corte raso. Para o isolamento dos fungos foi desenvolvido um meio de cultura de serragem de eucalipto-ágar. Dentre 292 isolados obtidos e submetidos ao teste de Bavendamm, 144 foram classificados como causadores de podridão-branca, capazes de produzir fenoloxidases. Dentre as nove relações C/N testadas, observou-se uma tendência de ocorrer maior degradação de cavacos naquelas iguais a 60 : 1, 200 : 1 e 300 : 1. Utilizando a relação C/N igual a 60 : 1, realizaram-se dois experimentos para avaliar a degradação de cavacos de Eucalyptus saligna por isolados fúngicos de podridão-branca. No primeiro experimento, avaliado aos 90 dias de incubação, foram selecionados sete isolados, que causaram perda de peso em cavacos superior ou igual à causada por Trametes versicolor, usado para comparação. No segundo experimento foram testados 46 isolados fúngicos. Dentre os mais eficientes estavam os sete isolados selecionados no primeiro teste, além de outros quatro isolados. Baseado na análise de DNA, seis isolados foram identificados, sendo três pertencentes à espécie Pycnoporus sanguineus, um ao gênero Peniophora sp., um ao gênero Pestalotiopsis sp. e um ao gênero Ganoderma sp.

RESISTÊNCIA BIOLÓGICA E COLORIMETRIA DA MADEIRA TERMORRETIFICADA DE DUAS ESPÉCIES DE EUCALIPTO1

RESUMO Os objetivos deste estudo foram os seguintes: a) observar o efeito de termorretificação sobre a durabilidade natural da madeira de Eucalyptus tereticornis e Corymbia citriodora após ataque de fungos causadores de podridão-branca (Trametes versicolor eGanoderma applanatum); b) avaliar parâmetros colorimétricos após a submissão das madeiras a tratamentos térmicos e aos fungos T. versicolor e G. applanatum. Para realização do estudo, corpos de prova de ambas as espécies foram submetidos aos tratamentos: T1 - testemunha; T2 - autoclave (120 °C) a 1,5 kgf/cm2 durante 1 h; T3 - estufa laboratorial (180 °C) por 4 h; e T4 - térmico combinado [120 °C (1 h) + 180 °C (4 h)]. Posteriormente, os corpos de prova foram submetidos ao apodrecimento acelerado, sendo expostos às duas espécies de fungos por 16 semanas. Após esse período, foram avaliadas a perda de massa e as variáveis colorimétricas, através dos parâmetros L*, a*, b*, C* e hº antes e depois do ataque pelos fungos. De acordo com os resultados, E. tereticornis e C. citriodora foram classificados como altamente resistentes a fungos apodrecedores, exceto no tratamento testemunha de C. citriodora submetido ao ataque de G. applanatum, o qual foi classificado como resistente. Com os tratamentos T3 e T4, a perda de massa foi reduzida em ambas as espécies de madeira. As maiores mudanças nos parâmetros colorimétricos ocorreram devido ao tratamento térmico e à pouca variação observada com o ataque dos fungos apodrecedores. Houve redução da variável claridade (L*) e queda das matrizes vermelho (a*) e amarelo (b*), razão por que o tratamento térmico mostrou-se como alternativa para proteção e escurecimento da madeira de eucalipto, tornando-a mais próxima de padrões de coloração de madeiras nobres.