Grape stalks as substrate for white rot fungi, lignocellulolytic enzyme production and dye decolorization.

The aim of this work was to evaluate the potential of grape stalks, an agroindustrial waste, for growth and lignocellulolytic enzyme production via solid-state fermentation, using the following three white rot fungi: Trametes trogii, Stereum hirsutum and Coriolus antarcticus. The decolorization of several dyes by the above mentioned cultures was also investigated. Similar values of dry weight loss of the substrate were measured after 60 days (33-43 %). C. antarcticus produced the highest laccase and Mn-peroxidase activities (33.0 and 1.6 U/g dry solid). The maximum endoglucanase production was measured in S. hirsutum cultures (10.4 U/g), while the endoxylanase peak corresponded to T. trogii (14.6 U/g). The C. antarcticus/grape stalk system seems potentially competitive in bioremediation of textile processing effluents, attaining percentages of decolorization of 93, 86, 82, 82, 77, and 58% for indigo carmine, malachite green, azure B, remazol brilliant blue R, crystal violet and xylidine, respectively, in 5 h.

Layer-by-layer self-assembled osmium polymer-mediated laccase oxygen cathodes for biofuel cells: the role of hydrogen peroxide.

High potential purified Trametes trogii laccase has been studied as a biocatalyst for oxygen cathodes composed of layer-by-layer self-assembled thin films by sequential immersion of mercaptopropane sulfonate-modified Au electrode surfaces in solutions containing laccase and osmium-complex bound to poly(allylamine), (PAH-Os). The polycation backbone carries the Os redox relay, and the polyanion is the enzyme adsorbed from a solution of a suitable pH so that the protein carries a net negative charge. Enzyme thin films were characterized by quartz crystal microbalance, ellipsometry, cyclic voltammetry, and oxygen reduction electrocatalysis under variable oxygen partial pressures with a rotating disk electrode. New kinetic evidence relevant to biofuel cells is presented on the detection of traces of H(2)O(2), intermediate in the O(2) reduction, with scanning electrochemical microscopy (SECM). Furthermore the inhibitory effect of peroxide on the biocatalytic current resulted in abnormal current dependence on the O(2) partial pressure and peak shape with hysteresis in the polarization curves under stagnant conditions, which is offset upon stirring with the RDE. The new kinetic evidence reported in the present work is very relevant for the operation of biofuel cells under stagnant conditions of O(2) mass transport.

Stabilization studies of Fomes sclerodermeus laccases.

Stability of laccase isoenzymes from a crude extract obtained from Fomes sclerodermeus grown on wheat bran medium was studied. The variables assessed were temperature, pH and additives. As revealed by PAGE, three bands of laccase, each with different thermal inactivation pattern, were detected in the crude extract: after 6h at 50 degrees C and pH 8, Lc2 was the most resistant, while the Lc1 and Lc3 bands were almost completely inactivated. This pattern of inactivation was observed at all temperatures and pH tested. Laccase activity was more stable in the 5-10 pH range when incubated at 40 and 50 degrees C; at 30 degrees C and 24h the enzyme remained fully active in the 3-11 pH range. The effect of additives (veratryl alcohol, trehalose, glycerol, mannitol, glutaraldehyde, CuSO(4) and 1-HBT) on laccase stability was tested. The stability was enhanced with CuSO(4) (1.25 mM), glycerol (0.2%) and mannitol (1%). The presence of both CuSO(4) and glycerol caused a 3-fold increase in the half-life values.

[Stereum hirsutum (Wild) Pers. action in dye degradation]. / Acción de Stereum hirsutum (Wild) Pers. en la degradación de colorantes.

Stereum hirsutum, a white rot fungus, has a good growth in solid state fermentation. This was carried on with wheat bran, soy bran and a mixture of both. Mycelia grown on soy bran showed the highest decolorization activity on Ponceau 2R (xylidine), indigo carmine and malachite green. Optimal relationship between decolorization and detoxification of malachite green was 30 g of fresh weight (mycelia plus substrate) in 500 ml malachite green solution, 42 U/l of laccase was measured in this solution. Decolorization was carried on without the addition either of nutrients or mediators. Conditions for maximal decolorization did not agree with those for maximal ligninolytic enzyme production, but effectiveness of laccase activity on decolorization was evidenced by electrophoretic analysis, that allowed laccase identification and its decolorization activity in gels stained with indigo carmine and malachite green, with ABTS as mediator. Detoxification was assayed using the sensible fungus Phanerochaete chrysosporium.

Modification of malachite green by Fomes sclerodermeus and reduction of toxicity to Phanerochaete chrysosporium.

Malachite green (MG) is a triphenylmethane dye used as a fungicide but also possesses a high toxicity to mammalian cells. The toxicity of MG to Fomes sclerodermeus and Phanerochaete chrysosporium was assessed. P. chrysosporium was highly sensitive to the dye and it was unable to grow on solid media containing 64 microM of MG, lower concentrations caused a delay in growth. The radial growth of F. sclerodermeus was not affected at this concentration and up to 128 microM. In liquid media both fungi were more sensitive. F. sclerodermeus not only was able to grow in the presence of high concentrations of MG, but also it was able to decolorize and detoxify the dye. MG treated with supernatants containing high laccase activity in the presence or absence of 1-hydroxybenzotriazole (1-HBT) gave a colorless product (DMG) that was not toxic to P. chrysosporium and other white rot fungi tested. On the basis of the data of maximal absorbance, it is probable that the mechanism involved in the modification of the dye was different if 1-HBT was added to the reaction.

[Combined effect of copper and initial pH of the culture on production of laccase and manganese peroxidase by Stereum hirsutum (Willd) Pers]. / Efecto combinado del cobre y pH inicial del medio de cultivo sobre la producción de lacasa y manganeso peroxidasa por Stereum hirsutum (Willd) Pers.

Stereum hirsutum is a white-rot fungus which produces laccase and manganese peroxidase as part of its ligninolytic system. Maximal ligninases production (under the conditions studied) was obtained in the media containing 250 microM Cu++ along with a pH value of 5.5. The fitting of ligninase production to a linear equation showed negative interaction between increasing values of pH and concentration of copper.

[Degradation of poplar wood by Fomes sclerodermeus: production of ligninolytic enzymes in sawdust of poplar and cedar]. / Degradación de madera de álamo por Fomes sclerodermeus: producción de enzimas ligninolíticas en aserrín de álamo y cedro.

Fomes sclerodermeus is a white-rot fungus. Its production of laccase, manganese peroxidase and lignin peroxidase on sawdust-based media was evaluated. No lignin peroxidase activity was measured in any media tested. The higher production of laccase and manganese peroxidase were found on media containing poplar sawdust. F. sclerodermeus was grown on wood blocks of poplar during six months. Dry weight losses of the blocks reached a mean value of 51%. The quantification of cellulose and lignin in the 6-months incubated blocks showed losses of up to 58 and 56% for cellulose and lignin, respectively. The decay examined under microscope revealed mycelium colonizing the lumen of vessel elements, cell wall thinning and entire degradation of the radial parenchyma.

[Enzymatic activity and degradation of different kinds of organic wastes by Saccobolus saccoboloides (Fungi, Ascomycotina)]. / Actividad enzimática y degradación de diferentes tipos de residuos orgánicos por Saccobolus saccoboloides (Fungi, Ascomycotina).

The ability to degrade organic solid wastes by the fungus Saccobolus saccoboloides was studied. The organism, unusual in such studies, was cultivated in synthetic liquid media with agitation, and on day 8 of growth the mycelium was passed to flasks with trimming. On day 16 of growth, the trimming degradation was assesed by carboxymethylcellulase, xylanase, and amylase activities evaluation, and NaOH 1% hydrolysis. Later on, the type of waste was modified (trimming, filter paper, newspaper, cardboard, sawdust and wood shaving were used) as well as the mass (300-1800 mg/flask). In these cases the enzymatic activities increased between 300 and 600 mg/flask. The total separation of the cellular components in all types of paper and cardboard was observed, together with a high loss of weight. S. saccoboloides was not able to degrade the wood wastes

Grape stalks as substrate for white rot fungi, lignocellulolytic enzyme production and dye decolorization / Uso del escobajo como sustrato para el crecimiento de hongos de la pudrición blanca, la producción de enzimas ligninolíticas y la decoloración de tinturas

The aim of this work was to evaluate the potential of grape stalks, an agroindustrial waste, for growth and lignocellulolytic enzyme production via solid-state fermentation, using the following three white rot fungi: Trametes trogii, Stereum hirsutum and Coriolus antarcticus. The decolorization of several dyes by the above mentioned cultures was also investigated. Similar values of dry weight loss of the substrate were measured after 60 days (33-43 %). C. antarcticus produced the highest laccase and Mn-peroxldase activities (33.0 and 1.6 U/g dry solid). The maximum endoglucanase production was measured in S. hirsutum cultures (10.4 U/g), while the endoxylanase peak corresponded to T. trogii (14.6 U/g). The C. antarcticus/grape stalk system seems potentially competitive in bioremediation of textile processing effluents, attaining percentages of decolorization of 93, 86, 82, 82, 77, and 58 % for indigo carmine, malachite green, azure B, remazol brilliant blue R, crystal violet and xylidine, respectively, in 5 h.

El objetivo de este trabajo fue evaluar el potencial del escobajo, un residuo agroindustrial, como sustrato para el crecimiento y la producción de enzimas lignocelulósicas de tres hongos causantes de pudrición blanca en la madera: Trametes trogii, Stereum hirsutum y Coriolus antarcticus. Para ello se utilizaron técnicas de fermentación en estado sólido. También se ensayó la decoloración de colorantes industriales sobre estos cultivos. La pérdida de peso seco del sustrato fue similar después del día 60 (33-43 %). C. antarcticus produjo las mayores actividades de lacasa y Mn-peroxidasa (33,0 y 1,6 U/g peso seco). La mayor actividad endoglucanasa fue medida en cultivos de S. hirsutum (10,4 U/g), y la mayor actividad endoxilanasa en T. trogii (14,6 U/g). El sistema C. antarcticus/escobap mostró un importante potencial para su aplicación en la biorremediación de efluentes textiles, con porcentajes de decoloración de 93, 86, 82, 82, 77 y 58 % para índigo carmín, verde de malaquita, azure B, azul R brillante de remazol, cristal violeta y xilidina, respectivamente, en 5 h.

Optimization of manganese peroxidase and laccase production in the South American fungus Fomes sclerodermeus (Lév.) Cke.

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu(2+), Mn(2+) and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn(2+), which acts as an inducer. Under these conditions Cu(2+) negatively affected MnP activity. At 13 days of growth 0.75 U ml(-1) were produced in the optimized culture medium supplemented with 1 mM MnSO(4) and 4 g l(-1) asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml(-1) in the presence of 0.2 mM CuSO(4), 0.4 mM MnSO(4) and 6 g l(-1) asparagine. Mn(2+) promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu(2+) and asparagine.