Introduction: Free-living amoebae (FLA) are unicellular organisms that live independently in the environment and are frequently present in wastewater. They play an important role in wastewater treatment by contributing to the degradation of organic matter and the regulation of bacterial populations, although certain species can cause opportunistic infections in humans. The most common genera include Vermamoeba, Acanthamoeba, Naegleria, and Vannella. Aim: The objective of this master’s thesis was to investigate the occurrence of individual genera of FLA at different stages of municipal wastewater treatment in two treatment plants (A and B) employing distinct treatment technologies. At wastewater treatment plant B, the process was complemented by an additional pilot-scale algal treatment system. Methods: A total of 22 water samples were collected from various treatment stages in the two plants during the spring seasons of 2024 and 2025. After isolation of FLA on non-nutrient agar with Escherichia coli as a food source, phenotypic identification was performed microscopically using Page’s identification key. For genotypic identification, DNA was extracted from amoebic cultures and subjected to polymerase chain reaction (PCR) with universal primers for FLA, followed by specific primers targeting the genera Acanthamoeba, Naegleria, Vermamoeba, and Vannella. Amplification products were confirmed by gel electrophoresis. Results: Phenotypic identification confirmed the presence of FLA in 21 out of 22 samples (95.5%), while genotyping revealed their presence in all samples. The most frequently detected genera were Naegleria (in approximately 90% of samples) and Vermamoeba (around 80%), whereas Acanthamoeba and Vannella were less common. A higher number of genera were detected in the supernatant compared to the sediment fraction of the prepared samples. The occurrence of FLA was high at the plant inlets (76%), decreased slightly in the primary sedimentation or mechanical treatment stage (72%), peaked during the biological (aerobic) phase and in the secondary clarifier/activated sludge reservoir (87.5%), and declined in the final effluent (56%). Their presence in the algal pond was comparable to that observed during mechanical treatment. Discussion and Conclusion: Although treatment plants A and B differ in their treatment technologies, both exhibited comparable FLA occurrence, highlighting the high resistance and adaptability of these organisms. The findings indicate that conventional treatment processes do not completely eliminate FLA, which is of particular concern for public health. FLA in wastewater treatment plants may act as reservoirs and vectors for opportunistic pathogenic bacteria such as Legionella and Mycobacterium. To mitigate environmental and human health risks, the implementation of additional disinfection methods (e.g., membrane filtration) and the inclusion of FLA as indicators of microbiological safety in water reuse practices would be advisable.
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