Evaluation of the genotoxic potential of reactive black 5 solutions subjected to decolorizing treatments by three fungal strains.

The genotoxic potential of solutions of the textile dye "Reactive Black 5" that were subjected to decolorizing treatments with the fungal strains Coriolopsis polyzona MUCL33483, Penicillium sp. MUBA001 and Pycnoporus sp. MUBA002 was tested. The genotoxicity of the solutions was determined by evaluation of micronuclei formation in Vicia faba root cells and calculation of a damage index (MN(ID)). Non-treated Reactive Black 5 solutions (50-1000 ppm) caused a statistically significant increase in micronuclei formation and, by then, in damage index. Solutions of dye treated with C. polyzona MUCL33483 and Pycnoporus sp. MUBA002 showed color loss, probably due to enzymatic breakdown of the colorant, but maintenance or even an increase in genotoxicity. On the other hand, the Penicillium sp. strain MUBA001 caused decolorization of the dye, apparently by adsorption on mycelia, and, for solutions that initially contained 50 ppm of colorant, an elimination of the genotoxicity was observed after three weeks of treatment.

Monitoring the active conformation of green fluorescent protein (GFP) and ß-glucosidase adsorbed on soil particles.

In order to determine the effect of various soil components on the activity of proteins, we monitored the fluorescence and the enzymatic activity of, respectively, green fluorescent protein (GFP) and ß-glucosidase adsorbed on fine soil particles. We also monitored the activity of these proteins in the presence of components that are representative of soil colloids: a montmorillonite clay, goethite and organic matter extracted from soil. Upon adsorption on clay and goethite, GFP lost its fluorescence properties while ß-glucosidase suffered only a partial loss of its catalytic activity. Extractable organic matter had an inactivating role on GFP while it did not cause inactivation of ß-glucosidase. When GFP and ß-glucosidase adsorbed on particles from natural soil samples, their behaviour was consistent with the behaviour observed for these proteins in the presence of the separate components, suggesting that the macroscopic activity of proteins adsorbed on soil particles corresponds to an average of the activities of proteins adsorbed on a mixture of surfaces. The monitoring of the proteins on soil particles with different organic matter contents has also shown that organic matter can have different effects (protecting or inactivating) on different proteins.

Fate of amyloid fibrils introduced in wastewater sludge.

Laboratory data on the behaviour of the pathogenic form of the prion protein (PrP(Sc)) in environmental matrices such as sewage sludge is scarce. Direct experiments with this misfolded protein require strict safety measures, pathogen class-3 facilities and costly reagents. However, preliminary data can be generated by non-pathogenic model systems that involve lower costs and simpler manipulation. We chose amyloid-like fibrils formed from the well-studied protein lysozyme as a model because, in the case of an accidental contamination of sewage sludge, PrP(Sc) would most likely be in the form of amyloid fibrils. All amyloid fibrils have similar structural features and tend to bind to thioflavin-T, thereby enhancing the fluorescence yield of the dye. We used this fluorescence enhancement to monitor amyloid fibrils introduced into activated sludge. We observed that, in the presence of sludge flocs, the concentration of amyloid fibrils that are detectable through the enhancement of thioflavin-T fluorescence decreased as a function of time, most likely due to hydrolysis of the fibrils by sludge proteases. Some of the fluorescence loss seems also due to the binding of sludge exopolymers to amyloid fibrils.

Factors affecting the fate of active proteins introduced in wastewater sludges: investigation with green fluorescent protein.

Green fluorescent protein (GFP) was used as a reporter protein to investigate the interactions and fate of the active conformation of proteins in wastewater sludge. GFP was chosen because its fluorescence is dependent on the integrity of its native conformation. We identified factors that cause the loss of GFP fluorescence when this protein is introduced in aerobic or anaerobic sludge. In both systems, soluble polymers present in the liquid fraction caused an initial loss of fluorescence, but stabilized the remaining fluorescent GFP molecules. In aerobic sludge, interaction of GFP with the sludge solids initially caused an additional loss of fluorescence but the remaining fluorescence kept fairly constant over the following hours. In anaerobic sludge, on the contrary, interaction of GFP with the sludge solids caused a temperature dependent loss of fluorescence, probably related to the presence of precipitated ferrous sulfide. No direct relationship was found between sludge protease activity and loss of GFP fluorescence, suggesting that proteases are not the primary factor controlling the fate of the active form of proteins in wastewater sludges.