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Doktorat (10.22Mb) UNIVERZITET U BEOGRADU RUDARSKO - GEOLOŠKI FAKULTET Vladimir S. Šaraba MIKROORGANIZMI – BIOHIDROGEOLOŠKI INDIKATORI ODABRANIH POJAVA MINERALNIH VODA SRBIJE doktorska disertacija Beograd, 2021. UNIVERSITY OF BELGRADE FACULTY OF MINING AND GEOLOGY Vladimir S. Šaraba MICROORGANISMS – BIOHYDROGEOLOGICAL INDICATORS OF SELECT MINERAL WATER OCCURRENCES IN SERBIA Doctoral Dissertation Belgrade, 2021 Mentori: Dr Veselin Dragišić, redovni profesor u penziji, Uža naučna oblast: hidrogeologija Univerzitet u Beogradu, Rudarsko-geološki fakultet Dr Ivica Dimkić, viši naučni saradnik, Uža naučna oblast: biologija mikroorganizama Univerzitet u Beogradu, Biološki fakultet Članovi komisije: Dr Vladimir Živanović, docent, Uža naučna oblast: hidrogeologija Univerzitet u Beogradu, Rudarsko-geološki fakultet Dr Jana Štrbački, docent, Uža naučna oblast: hidrogeologija Univerzitet u Beogradu, Rudarsko-geološki fakultet Dr Marina Ćuk Đurović, naučni saradnik, Uža naučna oblast: hidrogeologija Univerzitet u Beogradu, Rudarsko-geološki fakultet Dr Tamara Janakiev, naučni saradnik, Uža naučna oblast: biologija mikroorganizama Univerzitet u Beogradu, Biološki fakultet Dr Tanja Petrović Pantić, naučni saradnik, Uža naučna oblast: hidrogeologija Geološki zavod Srbije Datum odbrane:________________ Izjave zahvalnosti Najdublje izraze uvažavanja i veliko poštovanje iskazujem mentoru dr Veselinu Dragišiću, red. prof. RGF-a u penziji, koji je prepoznao moju istinsku želju za naučno-istraživačkim radom u oblasti biohidrogeologije, pod čijim je rukovodstvom uspešno realizovana predmetna doktorska disertacija. Zahvaljujem mu se na profesionalnom pristupu, maksimalnoj posvećenosti i nesebično prenesenom znanju tokom svih faza izrade doktorske disertacije. Podjednaku zahvalnost dugujem i mentoru dr Ivici Dimkiću, višem naučnom saradniku Biološkog fakulteta, koji me je uveo u „svet” mikroorganizama i omogućio da izvedem istraživanja podzemnih (mineralnih) voda Srbije sa trenutno najvećim dostignućima genotipske identifikacije mikroorganizama na osnovu 16S rDNK gena. Zahvaljujem se članovima Komisije, dr Jani Štrbački, docentu RGF-a i dr Tanji Petrović Pantić, naučnom saradniku Geološkog zavoda Srbije, na svim kompetentnim savetima tokom izrade doktorske disertacije. Dr Vladimiru Živanoviću, docentu RGF-a, dr Marini Ćuk Đurović, naučnom saradniku RGF-a i dr Tamari Janakiev, naučnom saradniku Biološkog fakulteta, iskazujem izraze zahvalnosti na učešću u Komisiji, budući da su prepoznali značaj i važnost doktorske disertacije u inženjerstvu podzemnih (mineralnih) voda. Dipl. biologu - specijalisti mikrobiologije Vesni Obradović i master mikrobiologu Tanji Vučković iz Instituta za vodoprivredu „Jaroslav Černi” izražavam zahvalnost na BART analizima ispitivanih uzoraka mineralnih voda, uz posebne izraze zahvalnosti gđi Vesni Obradović na izuzetno uspešnoj i produktivnoj saradnji od početka doktorskih akademskih studija pa sve do danas. Zahvalnost dugujem i dr Violeti Gajić, van. prof. i pokojnom doc. dr Predragu Vuliću sa Departmana za mineralogiju, kristalografiju, petrologiju i geohemiju RGF-a, koji su svojom predusretljivošću omogućili da uspešno kompletiram predmetnu doktorsku disertaciju. Dr Milojku Laziću, redovnom profesoru RGF-a u penziji, ukazujem dužno poštovanje na želji za razvojem hidrogeološke discipline biohidrogeologije, kojoj doktorska disertacija u najužem i osnovnom smislu pripada. Pokojnom dr Milanu Dimkiću, naučnom savetniku u penziji Instituta za vodoprivredu „Jaroslav Černi”, iskazujem izraze poštovanja na predusretljivom odnosu i kolegijalnom pristupu tokom učešća na Projektu TR 37014. Ministarstvu prosvete, nauke i tehnološkog razvoja Srbije izražavam naročitu zahvalnost na učešću u programu razvoja naučnoistraživačkih kadrova. Roditeljima, Slobodanu i Ranki, i sestri Jeleni dugujem bezgraničnu zahvalnost na podršci tokom višegodišnje edukacije na doktorskim akademskim studijama. U Beogradu, 2021. godine Vladimir Šaraba MIKROORGANIZMI – BIOHIDROGEOLOŠKI INDIKATORI ODABRANIH POJAVA MINERALNIH VODA SRBIJE Sažetak Sa namerom definisanja hidrogeološke uloge i značaja mikrobiološkog diverziteta odabranih pojava mineralnih voda Srbije, primenom sistema biodetektora, dokazano je prisustvo gvožđevitih, sulfato-redukujućih, sluz-produkujućih, heterotrofnih aerobnih i denitrifikacionih bakterija, na osnovu čega je proračunat rizik od razvoja procesa biokorozije i biohemijske inkrustacije, te zdravstveni rizik. Uz procenu rizika, izvedene su analize skenirajuće elektronske mikroskopije biofilmova i hidrogeohemijsko modeliranje, te makroskopska, otpička i difrakciona ispitivanja naslaga inkrustacije, dok je fizičko-hemijskim analizama utvrđena vrednost ukupne mineralizacije iznad 1 g/L, kao i povišeni sadržaji odgovarajućih metala i nemetala. Metabarkoding analizama mikrobioma mineralnih voda dokazano je prisustvo razdela Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes i Acidobacteria u većem procentu. Pseudomonas, Limnobacter i Rheinheimera su bili najzastupljeniji rodovi u uzorku mineralnih voda Torde, Acinetobacter i Massilia u uzorku iz Obrenovačke Banje, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis i Sulfuritalea u uzorku iz Lomnice, Pseudorhodobacter i Limnohabitans u mineralnim vodama Velike Vrbnice, dok su u uzorku iz Slankamen Banje dominirale zelene sumporne bakterije, Methylotenera, Flavobacterium, Melioribacter i Dechloromonas. Realizovanim istraživanjima zaključeno je da odgovarajući predstavnici mikroorganizama utiču na starenje bunara i definišu ekološki status vodnih resursa, dok s druge strane pojedini rodovi poseduju biotehnološki potencijal u tretmanu remedijacije kontaminiranih voda, zbog čega postoji neophodnost implementacije biohidrogeologije u okviru standardne metodike hidrogeoloških istraživanja. Ključne reči: biohidrogeologija, SEM analize, makroskopska i optička ispitivanja, rendgenska difrakcija praha, hidrogeohemijsko modeliranje, BART analize, metabarkoding analize mikrobioma, fizičko-hemijske analize, starenje bunara (biokorozija, biohemijska inkrustacija i biozarastanje), zdravstveni rizik. Naučna oblast: Geološko inženjerstvo Uža naučna oblast: Hidrogeologija UDK: 553.7:579.68(497.11)(043.3) MICROORGANISMS – BIOHYDROGEOLOGICAL INDICATORS OF SELECT MINERAL WATER OCCURRENCES IN SERBIA Abstract The presence of iron related, sulphate reducing, slime producing, heterotrophic aerobic and denitrifying bacteria was proved applying biodetectors, in order to define the hydrogeological role and importance of the microbiological diversity of select occurrences of mineral waters in Serbia. Then the biocorrosion, biochemical encrustation and health risks were assessed. The research included scanning electron microscopy of biofilms, hydrogeochemical modelling and macroscopic, optical and diffraction examination of the encrustations. Physical and chemical analyses indicated total dissolved salts of more than 1 g/L and elevated concentrations of certain metals and non-metals. Metabarcoding of the microbiomes of the mineral waters revealed the presence of a high percentage of the Proteobacteria, Bacteroidetes, Patescibacteria, Chlorflexi, Firmicutes, Spirochaetes and Acidobacteria divisions. Pseudomonas, Limnobacter and Rheinheimera were the most representative genera in the mineral water sample from Torda, Acinetobacter and Massilia in the sample collected in Obrenovačka Banja, Maritimimonas, Phreatobacter, Planktosalinus, Acinetobacter, Geminocystis and Sulfuritalea in the sample from Lomnica, and Pseudorhodobacter and Limnohabitans in the sample from Velika Vrbnica, while in the sample from Slankamen Banja was dominated by green sulphur bacteria, Methylotenera, Flavobacterium, Melioribacter and Dechloromonas. The research concludes that the respective microorganisms cause water well aging and define the ecological status of the water resources, while, on the other hand, some of the genera possess the biotechnological potential for treating and remediating contaminated waters, which is a compelling reason for implementing biohydrogeology in the standard hydrogeological research methodology. Keywords: biohydrogeology, SEM analyses, macroscopic and optical assessment, X-ray powder diffraction, hydrogeochemical modelling, BART analyses, metabarcoding of microbiomes, physical and chemical analyses, well aging (biocorrosion, biochemical encrustation and biofouling), health risk. Scientific field: Geological Engineering Scientific subfield: Hydrogeology UDC number: 553.7:579.68(497.11)(043.3) SADRŽAJ 1. UVOD .................................................................................................................................................................... 1 1.1 POLAZNE HIPOTEZE ........................................................................................................................... 3 1.1.1 Mikroorganizmi u podzemnim (mineralnim) vodama .................................................. 5 1.1.1.1 Mikroorganizmi u mineralnim vodama .................................................................... 15 1.1.1.2 Uloga i značaj poznavanja mikroorganizama u inženjerstvu podzemnih (mineralnih) voda ............................................................................................................................ 21 1.1.1.3 Patogeni i koliformni mikroorganizmi u podzemnim (mineralnim) vodama ................................................................................................................................................................
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