Susana Yagüe

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.

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.

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.