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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.

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.