20.500.12556/RUL-174470 Pojavnost prostoživečih ameb v različnih stopnjah čiščenja odpadne vode v dveh komunalnih čistilnih napravah magistrsko delo Occurrence of free-living amoebae in different stages of wastewater treatment in two municipal wastewater treatment plants master thesis Uvod: Prostoživeče amebe so enocelični organizmi, ki samostojno živijo v okolju in so pogosto prisotni tudi v odpadni vodi. Pri čiščenju odpadne vode imajo pomembno vlogo pri razgradnji organskih snovi in uravnavanju bakterijskih populacij, nekatere vrste lahko povzročajo oportunistične okužbe pri ljudeh. Najpogostejši rodovi so Vermamoeba, Acantamoeba, Naegleria in Vannella. Namen: Namen magistrskega dela je bil preučiti pojavnost posameznih rodov prostoživečih ameb v posameznih stopnjah čiščenja komunalne odpadne vode, v dveh komunalnih čistilnih napravah (A, B), z različnima tehnologijama čiščenja. Pri komunalni čistilni napravi B je v proces čiščenja vključen še dodatni pilotni alternativni sistem z algami. Metode dela: V dveh čistilnih napravah smo v različnih stopnjah čiščenja v spomladanskih obdobjih 2024 in 2025 skupno odvzeli 22 vzorcev vode. Po osamitvi prostoživečih ameb na nehranljivem gojišču z bakterijo E. coli smo jih fenotipsko opredelili pod mikroskopom s pomočjo Pagejevega identifikacijskega ključa. Za genotipsko identifikacijo smo iz kultur ameb ekstrahirali molekulo DNA in izvedli verižno reakcijo s polimerazo z univerzalnimi oligonukleotidnimi začetniki za prostoživeče amebe ter nato še s specifičnimi začetniki za rodove Acanthamoeba, Naegleria, Vermamoeba in Vannella. Dobljene pomnožke smo preverili z gelsko elektroforezo. Rezultati: Fenotipsko smo prostoživeče amebe po gojenju na gojiščih potrdili v 21 od 22 vzorcev (95,5 %), z genotipizacijo pa v vseh vzorcih. Najpogostejši rodovi so bili Naegleria (v približno 90 % vzorcev) in Vermamoeba (okoli 80 % vzorcev), medtem ko sta bila Acanthamoeba ter Vannella redkejša. Večje število preiskovanih rodov smo našli v supernatantu kot v sedimentu pripravljenega vzorca. Pogostost prostoživečih ameb je bila v vzorcih iz vtoka v čistilno napravo pogosta (76 %), nato je rahlo upadla v primarnem usedalniku oziroma po mehanskem čiščenju (72 %), dosegla vrh v biološki (aerobni) fazi in v sekundarnem usedalniku oziroma v zalogovniku z aktivnim blatom (87,5 %) ter se znižala v končnem iztoku (56 %). Njihova prisotnost v algnem bazenu je bila podobna kot v stopnji mehanskega čiščenja. Razprava in zaključek: Čeprav se čistilni napravi A in B razlikujeta po tehnologiji čiščenja, sta izkazovali primerljivo pojavnost prostoživečih ameb, kar potrjuje visoko odpornost ter prilagodljivost teh organizmov. Ugotovili smo, da standardni postopki čiščenja ne odstranijo vseh ameb, kar je posebej pomembno z vidika javnega zdravja. Amebe lahko v čistilnih napravah delujejo kot rezervoarji in prenašalci oportunistično patogenih bakterij, kot sta Legionella ter Mycobacterium. Za zmanjšanje tveganja za okolje in zdravje ljudi bi bilo smiselno uvesti dodatne metode dezinfekcije (npr. membranska filtracija) ter vključiti prostoživeče amebe kot indikatorje mikrobiološke varnosti pri ponovni uporabi vode. 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. magistrska dela sanitarno inženirstvo prostoživeče amebe komunalna odpadna voda stopnje čiščenja master's theses sanitary engineering free–living amoebae municipal wastewater treatment stages true false false [K. Slak] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-10-03 07:15:20 2025-10-03 07:15:50 2025-11-23 03:54:35 0000-00-00 00:00:00 2025 0 Ljubljana 0 Ljubljana 58 str. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 614 153093 251579395 54902.pdf 54902.pdf 1 E2CAF7E650C7C8F2A77BD6CFB66CEAE9 70d88f3255529586d96348668ce9984655d8667008c6b90825098f6be72ccb71 d6a8ddf6-a017-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=219282 Zdravstvena fakulteta 0 0 0