Extraction and characterization of polygalacturonase of Fomes sclerodermeus produced by solid-state fermentation / Extracción y caracterización de poligaracturonasa de Fomes sclerodermeus producida por fermentación en estado sólido

Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 °C and had optimum activity at pH 5 and 50 °C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.

Se estudió la producción de poligalacturonasa (PG) por Fomes sclerodermeus usando técnicas de fermentación en estado sólido. La actividad PG máxima (26 U/g ps) fue observada entre los días 11 y 13. La actividad PG en los extractos crudos fue más estable a pH 5-6 y 30 °C, con una actividad óptima a pH 5 y a 50 °C. Las condiciones óptimas para la extracción de PG se lograron con una única extracción empleando Na2SO4 como solvente, con 10 minutos de agitación. En el escalado del sistema, la actividad PG por gramo de peso seco de sustrato disminuyó cerca de 60% comparada con la obtenida en frascos Erlenmeyer, pero la actividad total fue mayor.

Degradación de hojas de pino por Stereum hirsutum / [Degradation of pine needles by Stereum hirsutum]

Pine-needle degradation by Stereum hirsutum was studied under conditions of solid state fermentation with the aim of accelerating its decomposition, avoiding the accumulation in situ and in view of the possible utilization of the residual organic matter. Three experimental systems were tested: pine needles alone and with the addition of either a nitrogen source or barley grain. Determinations were made at 14 and 28 days of incubation. All treatments showed substrate degradation. The addition of a nitrogen source raised enzymatic activities measured but not the degree of degradation. Grain addition resulted in higher biomass, enzyme activities, sugar accumulation and degradation of the substrate. Fungal biomass estimated as N-acetyl glucosamine allowed calculation of the actual degradation of the substrate, that reached 19

at 28 d of culture without additions and 44

at 14 d in pine-needles with grain.

Degradation of environmental pollutants by Trametes trogii

The ability of the ligninolytic fungus Trametes trogii to degrade in vitro different xenobiotics (PCBs, PAHs and dyes) was evaluated. Either 200 ppm of a PCB mixture (Aroclor 1150) or 160 ppm of an industrial PAH mixture (10 V/V of PAHs, principal components hexaethylbenzene, naphthalene, 1-methyl naphthalene, acenaphthylene, anthracene, fluorene and phenanthrene), were added to trophophasic and idiophasic cultures growing in a nitrogen limited mineral medium (glucose/asparagine) and in a complex medium (malt extract/glucose). Gas-liquid chromatography proved that within 7 to 12 d more than 90 of the organopollutants added were removed. The decrease in absorbance at 620 nm demonstrated that cultures of this fungus were able to transform 80 of the dye Anthraquinone-blue (added at a concentration of 50 ppm) in 1.5 h. Enzyme estimations indicated high activity of laccase (up to 0.55 U/mL), as well as lower production of manganese-peroxidase. Laccase activity, detected in all the conditions assayed, could be implicated in the degradation of these organopollutants. Considering the results obtained, T. trogii seems promising for detoxification.

Efecto del cocultivo de especies fúngicas sobre la degradación de residuos lignocelulósicos / [Effect of cocultivating fungal species on the degradation of lignocellulose residues]

The degradation potential of Phanerochaete sordida, Trametes trogii, Coprinus truncorum and Paecilomyces sp. upon yard wastes was evaluated. The species had been inoculated individually or in pairs formed by P. sordida and Paecilomyces sp., T. trogii and Paecilomyces sp., and C. truncorum and Paecilomyces sp. The highest level of endoxilanase activity was produced by P. sordida growing alone, during day 21 (1.09 U/g of dry material), but in P. sordida and Paecilomyces sp. cultures, the detected activity did not overcome 0.27 U/g of dry material during the whole experiment. T. trogii showed maximum activity on day 14 (0.78 U/g of dry material), but in T. trogii and Paecilomyces sp. cultures, the values increased until day 21 (1.07 U/g of dry material). P. sordida endocellulase activity reached its maximum on day 28 (0.08 U/g of dry material), but in P. sordida and Paecilomyces sp. cultures, this activity increased during the whole experiment (0.04 U/g of dry material). The major weight loss was found in P. sordida (27.6

). The possible beneficial effect of co-culture in yard wastes biodegradation is discussed.

Degradación de residuos vegetales por Coprinus truncorum usando dos métodos de cultivo / Degradation of plant waste by Coprinus truncorum using 2 culture methods

Degradation of yard wastes by Coprinus truncorum growing in a vertical aereated bioreactor or in flasks was studied. There was a constant decay of reducing sugars in the medium that avoided their accumulation and their possible repression of degradative enzymes. Endoxylanase activity at first showed a similar pattern in both culture conditions, with maximal activity on the 12th day, but flasks maintained a high activity thereafter. Flasks also showed a higher endoglucanase activity with a peak on the 18th day, whereas the maximal value in the bioreactor was reached on the 26th day. No Mn-peroxidase and only low values of laccase activity were found. The measurements of pH and soluble proteins during the incubation period were suitable indicators of the degradation process by C. truncorum.

Degradación de azui de antraquinona por Trametes trogii / [Degradation of anthraquinone blue by Trametes trogii]

The ability of the white rot fungus Trametes trogii BAFC 463 (high producer of ligninolytic enzymes, especially laccase and manganese peroxidase) to degrade the dye anthraquinone blue, refractory to bacterial attack, was evaluated. Both tropho- and idiophasic T. trogii cultures in synthetic medium (glucose/asparagine) and complex medium (malt extract/glucose) were able to transform up to 88

dye in 4 hours. The activity of laccase, an oxygen-dependent phenoloxidase which was present at high levels in all the conditions assayed, might be related to the ability of the fungus to degrade the colorant. This is supported by the fact that in bioreactor experiences carried out at pH 4.5 the addition of anthraquinone blue caused a decrease in the levels of soluble oxygen. However, although high levels of laccase were produced at pH 7.5, the enzyme was not active, and neither dye transformation nor loss in the levels of soluble oxygen were quantified.

Cellulose and xylan degrading enzymes in Thecotheus pelletieri

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Enzimas de hongos de pudrición blanca involucradas en la degradación de lignina / Enzymes of white rot fungi involved in lignin degradation

The presence of oxidases and peroxidases was tested qualitatively in 12 strains of white rot Basidiomycetes. Plate tests with gallic acid, tannic acid, guayacol, Poly R-478 and Azure B were used. Fomes sclerodermeus, Phlebia sp. and Pycnoporus sanguineus were selected for further studies because they produced the largest areas of degradation in all media tested. Poly R-478 degradation and manganese peroxidase, lignin peroxidase and laccase activities were measured in glucose-asparagine (N-sufficient) and Kirk (N-limited) media. The highest activities were produced by F. sclerodermeus cultured in glucose asparagine medium.

Efecto de las condiciones de cultivo sobre la actividad ligninolÝtica de Stereum hirsutum / Influence of culture conditions on ligninolytic activity of Stereum hirsutum

Stereum hirsutum BAFC 2234 was tested for growth kinetics and ligninolytic enzyme production. The strain showed weak coincidences with widely studied organisms (Phanerochaete chrysosporium, Pleurotus ostreatus) when the response to nitrogen sources was assayed. This could be interpreted as a different regulation to nitrogen metabolism for S. hirsutum BAFC 2234. Alternative carbon sources and the addition of veratryl alcohol and wood extracts were also tested showing partial correspondence with other ligninolytic fungi. On the basis of the high enzymatic activities observed, S. hirsutum BAFC 2234 could be a suitable source of lignin degrading system aiming techological processes.

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.

Desarrollo de Iodophanus carneus ante distintas calidades de luz / [Development of Iodophanus carneus under different types of light]

Development of Iodophanus carneus was conditioned by light. Hyphal growth was photoinhibited. Light wavelength increases carotene contents being blue and near ultraviolet light the most effective. For ascocarp induction or inhibition, the most effective wavelengths were blue. The possible role of flavins is discussed.

Influencia de fuentes carbonadas y nitrogenadas sobre la actividad celulolítica de Trametes trogii / [Effect of carbon and nitrogen sources on the cellulolytic activity of Trametes trogii]

Trametes trogii was grown in a liquid synthetic medium containing different carbon and nitrogen sources. Enzymatic activities of cellulases (endoglucanase, exoglucanase and beta-glucosidase) were measured in culture supernatants. Organic nitrogen sources were the most favourable for growth and cellulase production. Increasing nitrogen concentrations also increased cellulase production. Among carbon sources, crystalline cellulose, cellobiose and a mixture of carboxymethylcellulose and cellobiose induced maximal endoglucanase production. The optimal concentration of the carbon source was 10 g/l.

Influencia de la luz sobre el desarrollo de Saccobolus platensis / [Effect of light on the development of Saccobolus platensis]

The ascomycetous fungi Saccobolus platensis requires light for apothecial production. Photoinduction could be accomplished by continuous light and also by minor light periods. When apothecia were photoinduced by single 24 hour photoperiods, the effect varied according to the stage of development of the mycelium: it was most photoreceptive at the 5th day of growth. At this stage a single 6 hour irradiation was enough to induce 50

fructifications of the control (continuous light). After photoinduction, protoapothecia reached maturity independently of light treatment. Light quality influenced both apothecial number and size. Near ultraviolet light was the most effective for the induction of apothecia, followed by blue light; the maximum apothecial size was reached under blue light.