María C. Terrón

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.

Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1

Summary The biology of caveolin-1 (Cav1)/caveolae is intimately linked to actin dynamics and adhesion receptors. Caveolar domains are organized in hierarchical levels of complexity from curved or flattened caveolae to large, higher-order caveolar rosettes. We report that stress fibers controlled by Abl kinases and mDia1 determine the level of caveolar domain organization, which conditions the subsequent inward trafficking of caveolar domains induced upon loss of cell adhesion from the extracellular matrix. Abl-deficient cells have fewer stress fibers, a smaller pool of stress-fiber co-aligned Cav1 and increased clustering of Cav1/caveolae at the cell surface. Defective caveolar linkage to stress fibers prevents the formation of big caveolar rosettes upon loss of cell adhesion, correlating with a lack of inward trafficking. Live imaging of stress fibers and Cav1 showed that the actin-linked Cav1 pool loses its spatial organization in the absence of actin polymerization and is dragged and clustered by depolymerizing filaments. We identified mDia1 as the actin polymerization regulator downstream of Abl kinases that controls the stress-fiber-linked Cav1 pool. mDia1 knockdown results in Cav1/caveolae clustering and defective inward trafficking upon loss of cell adhesion. By contrast, cell elongation imposed by the excess of stress fibers induced by active mDia1 flattens caveolae. Furthermore, active mDia1 rescues the actin co-aligned Cav1 pool and Cav1 inward trafficking upon loss of adhesion in Abl-deficient cells. Thus, caveolar domain organization and trafficking are tightly coupled to adhesive and stress fiber regulatory pathways.

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.

Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus

ABSTRACT Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain I-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain I-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.

Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62

Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins.

Molecular Characterization of Alkalilignin‐1M30C: a Valid Alternative Preparation to the Björkman Lignin in the Analytical Study of Wheat Straw Lignin

Bjorkman lignin, obtained by extensive grinding and dioxane extraction, is considered one of the most suitable standard preparations close to the native lignin from lignocellulosic materials. In this study, alkalilignin extracted with 1 M NaOH at 30 °C (AL-1M30C), and the Bjorkman lignin from wheat straw (Triticum aestivum) were compared using spectroscopic and degradative techniques. The very small chemical differences between the two preparations are mainly that the Bjorkman method yields a lignin with slightly higher proportion of p-hydroxyphenyl units, and some co-extracted aliphatic fractions. The AL-1M30C preparation, on the other hand, showed a somewhat higher proportion of syringyl units, as well as the probable effects of the selective removal of structures associated with ester carbonyl groups. The evident similarity between both lignin samples suggests that AL-1M30C produced from wheat straw may represent a convenient alternative to Bjorkman lignin preparation for analytical purposes. These results also point towards the possibility of producing suitable lignin preparations from other Gramineae plants showing high solubility in alkaline reagents.

Pyrolysis—gas chromatography/mass spectrometry of wheat straw fractions obtained by alkaline treatments used in pulping processes☆

Abstract Different wheat straw fractions representative of industrial and laboratory-scale alkaline pulping processes have been studied by pyrolysis—gas chromatography/mass spectrometry (Py-GC/MS). For comparative purposes, a milled straw lignin from wheat straw was also studied. The pyrolytic patterns were suitable for estimating the extent to which lignin-derived material is present in the hemicellulose preparations, or hemicellulose-derived products contributed to the alkalilignin fractions. The results reflect the different efficiency of the treatments to remove lignin and carbohydrate during alkaline pulping of Gramineae straws. The virtual lack of carbohydrate-derived pyrolysis products was observed only in the alkalilignin preparation obtained with the highest NaOH concentration studied (1 M) at 30°C for 12 h. The pyrolysis data showed that the guaiacyl fragments were dominant in the lignin associated to hemicellulose fractions, suggesting structural relationships between hemicellulose and lignin through guaiacyl type units. This was complementary to the large amounts of syringyl units that remained in the alkalilignin preparations. Although Py-GC/MS provides markers for the major classes of cell wall components, such as lignin and structural polysaccharides, it does not yield fragments which could be ascribed with certainty to cinnamic acids, which are abundant and play an important role in cereal straws as lignin—carbohydrate linkages.

Pyrolysis-gas chromatography-mass spectrometry characterization of wheat straw alkaline-cooking effluents after biological treatment with the fungi Phanerochaete chrysosporium and Ganoderma australe

Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) was applied in order to monitor the extent of chemical modification of lignin units after seven-day treatment of paper-industry wastewaters using two ligninolytic fungi. Py-GC-MS after the effluent treatment with Ganoderma australe revealed an increase in the lignin compounds, in which oxidation and shortening of the side chain of lignin molecules have occurred. In addition, no significant alkalilignin weight-loss nor reduction of lignin pyrolysis products were detected. On the other hand, these chemical transformations of lignin units were not observed after the effluent treatment with Phanerochaete chrysosporium. However, a remarkable reduction of lignin pyrolysis products and a large alkalilignin weight-loss were observed. The percentages of decolorization attained at day seven of culture were similar for both fungi. These results are possibly due to the different lignin-degrading mechanisms and/or to the earlier expression of the ligninolytic system of P. chrysosporium, under the experimental conditions assayed. Effluent decolorization could not be attributed to adsorption on the mycelia, since no significant quantity of alkalilignin was found attached to the mycelia of either fungi.

Identification of a new laccase gene and confirmation of genomic predictions by cDNA sequences of Trametes sp. I-62 laccase family

The strain Trametes sp. I-62 (CECT 20197) is a white-rot fungus with great potential for biotechnological applications in the fields of industrial waste water decolorization and clean up. Three laccase genes: lcc1, lcc2 and lcc3 have been cloned and sequenced from this basidiomycete. In this work, the coding regions of the corresponding cDNAs have been synthesized, cloned, and sequenced. They are 1563, 1563 and 1575 bp in length, respectively. Former putative intron/exon structures from genomic DNA are fully confirmed by match analysis with our cDNA sequences. Using Polymerase Chain Reaction--Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, an additional laccase cDNA was also identified, corresponding to a new gene, lcc1A, which displayed 99.6% identity with lcc1 at protein level. Such high similarity between lcc1 and lcc1A sequences, and the comparison with reports from other basidiomycete laccases, suggest that in this strain these two genes are allelic variants.