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Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.Over the last two decades, considerable work has been done with the goal of using microorganisms as bioremediation agents in the treatment of wastewater containing textile dyes. These contaminants contribute a minor fraction to the usually high load of dissolved organic matter in textile effluents (35), but they are highly visible and must be removed in order to comply with the regulations concerning effluent discharge.Azo aromatic dyes are the major group of textile dyestuffs. These structures can be reductively cleaved into colorless amines by several bacterial species (for reviews, see references 2, 9, 41, and 43); nevertheless, azo dye reduction occurring in the presence of living matter can be an abiotic process. An example of this is the reduction of acid orange 7 (called dye IV in the present study) and reactive red 2 in anaerobic sludge, where sulfide, produced by sulfate-reducing microorganisms, can reduce azo bonds (45). In most of the reported processes of azo dye bioreduction, however, the participation of an enzymatic activity is assumed. Since the products of azo dye reduction, with few exceptions (4,11,26), cannot be used by bacteria as carbon and energy sources, the cleavage of azo bonds is a gratuitous process which can occur when the microorganisms use a reduced carbon compound as the gro...

Biofilms are commonly involved in medical device-related infections. The purpose of this study was to determine the antimicrobial and anti-biofilm activity of polyethyleneimine (PEI) and PEIbased nanoparticles (nanoPEI) against Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii and Candida albicans (clinical and ATCC strains), and to evaluate their effect upon biofilm formation on polyurethane (PUR)-like catheters. MICs and minimal lethal concentrations of PEI and nanoPEI were determined according to CLSI microdilution reference protocols. For PEI, the MIC value was 195.31 mg l "1 for all the bacteria and 48.83 mg l "1 for the yeast strains. For nanoPEI, the MIC value was 1250 mg l "1 for all the strains except A. baumannii, for which it was 2500 mg l "1. Biofilm formation was assessed with PUR-like catheter segments and biofilm metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. Plasma membrane integrity and membrane potential were assessed by flow cytometry after staining microbial cells with a membrane-impermeable dye, propidium iodide, and a membranepotential marker, DiBAC 4 (3). PEI inhibited growth of all microbial species; higher concentrations of nanoPEI were needed to inhibit growth of all species. Biofilm formation in the presence of antibacterial PEI activity was dose-dependent (except for S. epidermidis) and species-related. NanoPEI at 0.5¾MIC and MIC significantly reduced the metabolic activity of biofilms of S. aureus, S. epidermidis and A. baumannii, whereas 2¾MIC was required in order to inhibit biofilm metabolic activity.

A number of anaerobic and aerobic bacterial species are known to decolourise azo dyes through the reduction of the azo bonds, forming the corresponding amines. In this work, we describe improved decolourisation conditions for model azo dyes by the ascomycete yeast Candida zeylanoides. The dyes were derived from the diazonium salts of metanilic and sulfanilic acids and N,N-dimethylaniline or 2-naphthol as coupling components. Total decolourisation times observed in culture media supplemented with 0.2mM dye ranged from 40 go 60 hours. The initial decolourisation rates were 14-52μmol.(g dry cell) -1 .h -1 , depending on dye structure. In the course of decolourisation either metanilic acid or sulfanilic acid were detected in the supernatant fluid, showing that decolourization by this yeast strain is due to azo bond reduction. None of those aminobenzenesulphonates supported microorganism growth as carbon and energy source but both could be used, to a limited extent, as nitrogen sources. The azo reductase activity is not significantly affected by pre-adaptation of the microorganism to the dyes.

Two biological approaches for decolorization of azo sulfonated dyes have been compared: reductive decolorization with the ascomycete yeast Issatchenkia occidentalis and enzymatic oxidative decolorization with Trametes villosa laccase alone or in the presence of the mediator 1-hydroxybenzotriazole. The redox potential difference between the biological cofactor involved in the reductive activity of growing cells and the azo dye is a reliable indication for the decolorization ability of the biocatalyst. A linear relationship exists between the redox potential of the azo dyes and the decolorization efficiency of enzyme, enzyme/mediator, and yeast. The less positive the anodic peak of the dye, the more easily it is degraded oxidatevely with laccase. The more positive the cathodic peak of the dye, the more rapidly the dye molecule is reduced with yeast.

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeastmediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae ⌬fre1 and ⌬fre1 ⌬fre2 mutant strains, but not the ⌬fre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae ⌬fre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.Research work on biodegradative processes for azo dyes usually exploits bacterial species, either isolated or in consortia (4, 36). Bacteria, under appropriate conditions (e.g., oxygen limitation and the presence of substrates utilized as carbon and energy sources), frequently reduce azo dyes, producing colorless amines. Nevertheless, many dyes are recalcitrant to conventional wastewater treatment processes with activated sludge (4). The overall impression in this research area is that many azo dyes can be reduced (and decolorized) by a considerable number of bacterial species, but as far as we know, the enzyme responsible for the unspecific primary reduction step has never been positively identified. What is currently postulated is that reductive decolorization of sulfonated azo dyes by living cells must occur extracellularly due to the impermeant nature of those compounds and that the primary reductant is a cytoplasmic electron donor, presumably NAD(P)H (36).Previous studies (30,31) have demonstrated that some nonconventional ascomycete yeasts are efficient azo dye decolorizers, acting, as many bacteria, by reducing the azo bond. Dye decolorization by yeasts is comparatively unspecific but is affected by the medium composition, by the yeast strain used, and by parameters such as pH and dissolved oxygen level. It also requires actively growing cells, being faster during the exponential growth phase, and displays an enzyme-like temperature profile, strongly suggesting its biotic nature. However, further information is required for successful application of yeasts in a wastewater treatment process. The present work was developed to demonstrate the participation of an externally directed plasma membrane redox system (PMRS) in azo dye reduction, linking an intracellular reductant to an extracellular electr...

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