Aldo E. González

The white-rot fungus Pleurotus ostreatus secretes laccase isozymes with different substrate specificities.

Four laccase isozymes (LCC1, LCC2, LCC3 and LCC4) synthesized by Pleurotus ostreatus strain V-184 were purified and characterized. LCC1 and LCC2 have molecular masses of about 60 and 65 kDa and exhibited the same pI value (3.0). Their N termini were sequenced, revealing the same amino acid sequence and homology with laccases from other microorganisms. Laccases LCC3 and LCC4 were characterized by SDS-PAGE, estimating their molecular masses around 80 and 82 kDa, respectively. By native isoelectrofocusing, their pI values were 4.7 and 4.5, respectively. When staining with ABTS and guaiacol in native polyacrilamide gels, different specificities were observed for LCC1/LCC2 and LCC3/LCC4 isozymes.

Pyrolysis/gas chromatography/mass spectrometry monitoring of fungal-biotreated distillery wastewater using Trametes sp. I-62 (CECT 20197)

Distillery wastewaters generated by ethanol production from fermentation of sugar-cane molasses, named vinasses, lead to important ecological impact due to their high content of soluble organic matter and their intense dark-brown color. Taking advantage of the well-known ability of white-rot fungi to degrade an extensive variety of organic pollutants, the capacity of Trametes sp. I-62 (CECT 20197) to detoxify this type of effluents was evaluated. In this work, pyrolysis/gas chromatography/mass spectrometry was applied to the chemical characterization of several fractions of Cuban distillery wastewater as well as to monitoring the changes which occurred after fungal treatment with this white-rot basidiomycete. Maximum effluent decolorization values and chemical oxygen demand reduction attained after seven days of fungal treatment were 73.3 and 61.7%, respectively, when 20% (v/v) of distillery vinasses was added to the culture medium. Under these conditions a 35-fold increase in laccase production by Trametes sp. I-62 was measured, but no manganese peroxidase activity could be detected. The pyrolysis/gas chromatography/mass spectrometry results showed a decrease in a number of pyrolysis products after seven days of fungal treatment, mainly furan derivatives. The decrease in the relative areas of these compounds could be related to the vinasse color-removal associated with melanoidin degradation. All these results indicated the potential use ofTrametes sp. I-62 in the detoxification of recalcitrant distillery vinasses.

Solid-state NMR studies of lignin and plant polysaccharide degradation by fungi

Solid-State NMR Studies of Lignin and Plant Polysaccharide Degradation by Fungi ByA.T. Martinez, A.E. Gonzalez*, M.Valmaseda*, B.E. Dale, MJ. Lambregts and J.E Haw ^Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Veläzquez 144, E-28006 Madrid, Spain Department of Chemical Engineering,Texas A & M University, College Station, Texas 77843-3122, U.S. A. Department of Chemistry,Texas A & M University, College Station,Texas 77843-3122, U.S. A.

Studies of the production and characterization of laccase activity in the basidiomycete coriolopsis gallica, an efficient decolorizer of alkaline effluents

Abstract The basidiomycete Coriolopsis gallica decolorizes alkaline paper effluents efficiently. In this work, we found that C. gallica produces laccase during this decolorization process. This enzymatic activity was produced in all media studied; however, the highest enzymatic activity was obtained in a medium containing paper effluent, where laccase was detected on the 2nd day of the experiment. The laccase activity of C. gallica was purified and characterized. The amino-terminal sequence of this protein showed the highest similarity with the laccase I of the basidiomycete PM1 and with Coriolus hirsutus laccase.

A study of yeasts during the delignification and fungal transformation of wood into cattle feed in Chilean rain forest

In Chilean rain forest the presence and distribution of yeasts were studied during the fungal transformation of wood, producing a partially delignified material used as cattle feed (“huempe”). A total of 68 yeast species was reported, including a noticeable proportions of basidiomycetous and xylose-assimilating strains, the latter related to the increased availability of this sugar in the decayed wood. The results of the mathematic comparison of taxonomic data of 327 strains showed a delimitation of sharp clusters corresponding to different species, including several new species described from this substrate, and genera, as Rhodotorula and Cryptococcus.At the initial stages of wood decay, characterized by the presence of Candida species, yeast diversity was low. The highest values were found in the medium stage of decay, used as feed, with an important presence of the genera Apiotrichum, Rhodotorula, Cryptococcus and Schizoblastosporion. After establishing a non-linear ordination from Euclidean distances between samples, based on yeast abundances, a relationship was observed between the populations developing at the different stages of wood decay by Ganoderma applanatum. A succession of yeast species was demonstrated during wood degradation by this white-rot fungus.

Kraft pulp biobleaching using an extracellular enzymatic fluid produced by Pycnoporus sanguineus.

The aim of this work was to obtain a LMS pre-treatment applicable to industrial TCF bleaching. Kraft pulp from Eucalyptus globulus was treated at 40 degrees C/pH 3 and 60 degrees C/pH 5 for 1h using an extracellular fluid enriched in laccase produced by Pycnoporus sanguineus and acetosyringone as mediator (HBT was used as a control mediator) (L). Alkaline extraction (E) and hydrogen peroxide (P) stages were then assayed. The LEP alternative was an efficient sequence to bleach kraft pulp since the enzymatic pre-treatment boosted the subsequent chemical bleaching. The best L pre-treatment was obtained with laccase-acetosyringone at 40 degrees C/pH 3. It reduces kappa number and hexenuronic acids, increases pulp viscosity, lowers hydrogen peroxide consumption down to an 87.4% (94.0% without L) and enhances brightness up to a 59% ISO (51% ISO without L).

Pyrolysis-gas chromatography/mass spectrometry of milled wood lignin of two Chilean woods naturally decayed by Ganoderma australe, Phlebia chrysocrea and a brown-rot fungus

Two native Chilean woods (Eucryptia cordifolia Cav. and Laurelia philippiana Looser) and their corresponding sound and degraded milled wood lignin preparations, were characterized by pyrolysis-gas chromatography/mass spectrometry. The main goal of this work was to study the chemical changes in a lignin molecule during the natural fungal decay. A higher proportion of syringyl (S) than guaiacyl (G) units was detected in the native wood of E. cordifolia (S/G ratio of 3.9), whereas L. philippiana native wood showed similar amounts of S and G units (S/G ratio of 0.9). The modified Bjorkman method used here proved to be a suitable procedure for removing almost all the carbohydrate fraction of wood, with only minor modifications (demethylations) to the resulting purified lignin. The alterations affecting the propane chain of lignin units (C 3 -alkyl chain oxidations and shortenings) observed in L. philippiana after Phlebia chrysocrea (Berk. and Curt.) decay were similar to these described in E. cordifolia by Ganoderma australe (Fr.), except that the former were only detected in S-lignin units. Additionally, in L. philippiana some demethylation reactions were also suggested, although oxidations were not observed in the C β -position. Both fungi produced a decrease in lignin content, independent of the different S/G ratio of the studied woods. After the brown-rot fungus attack the lignin content did not change. Although this brown-rot microorganism does not substantially degrade lignin, it is able to produce some minor chemical alterations: demethylations and oxidations of lignin side-chains

Biotreatment of tannin-rich beer-factory wastewater with white-rot basidiomycete Coriolopsis gallica monitored by pyrolysis/gas chromatography/mass spectrometry

Some fractions of beer-factory wastewaters represent an important environmental concern owing to their high content of polyphenols and dark-brown color. The capacity of Coriolopsis gallica to preferentially degrade lignin has been successfully applied in our laboratory to the biotreatment and decolorization of paper-industry effluents. In this work, the ability of this white-rot fungus to degrade high-tannin-containing wastewaters is evaluated. Under all the conditions studied, effluent decolorization and chemical oxygen demand reduction achieved by C. gallica at day 12 of incubation were close to 50 and 65%, respectively. No adhesion of dark color to the fungal mycelium was observed suggesting that decolorization could be ascribed to C. gallica degradation systems. Mycelium dry-weight values showed that C. gallica is tolerant to relatively high tannin content present in the effluent samples. In the sample containing the highest effluent concentration (60% v/v), dry-weight values suggested an inhibition of fungal growth at day 6 of incubation and a further adaptation of the fungus to the stressing tannin effect at day 12 of fungal treatment. Pyrolysis/gas chromatography/mass spectrometry results showed a decrease of polyphenols pyrolysis products, mainly phenol and guaiacol, with the incubation time. All these results indicate the potential use of C. gallica in bioremediation of tannin-containing industrial wastewaters and in other applications where a reduction in polyphenols content is required.

Photocatalytic elimination of indoor air biological and chemical pollution in realistic conditions

The photocatalytic elimination of microorganisms from indoor air in realistic conditions and the feasibility of simultaneous elimination of chemical contaminants have been studied at laboratory scale. Transparent polymeric monoliths have been coated with sol-gel TiO(2) films and used as photocatalyst to treat real indoor air in a laboratory-scale single-step annular photocatalytic reactor. The analytical techniques used to characterize the air quality and analyze the results of the photocatalytic tests were: colony counting, microscopy and PCR with subsequent sequencing for microbial quantification and identification; automated thermal desorption coupled to gas chromatography with mass spectrometry detection for chemical analysis. The first experiments performed proved that photocatalysis based on UVA-irradiated TiO(2) for the reduction of the concentration of bacteria in the air could compete with the conventional photolytic treatment with UVC radiation, more expensive and hazardous. Simultaneously to the disinfection, the concentration of volatile organic compounds was greatly reduced, which adds value to this technology for real applications. The fungal colony number was not apparently modified.

Laccase production by Pycnoporus sanguineus under different culture conditions.

Pycnoporus sanguineus is a white‐rot fungus that produces ligninolytic enzymes such as laccases. These enzymes can endure temperatures as high as 60 °C and are useful for pulp bleaching, dye decolorization and phenolic degradation.