MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF EMERGENCE AND ON WELLHEADS MINERALNE VODE V SRBIJI IN RAZVOJ FOTOTROFNIH MIKROBNIH ZDRUŽB NA MESTIH IZVIROV IN NA USTJIH VRTIN Vladimir ŠARABA1*, Slađana POPOVIć2, Olivera KRUNIć1, Gordana SUBAKOV SIMIć3, Željko KLJAJIć1 & Milojko LAZIć1 1 University of Belgrade, Faculty of Mining and Geology, Đušina 7, Belgrade, Serbia, e-mails: vladimirsaraba@ymail.com, olivera.krunic@rgf.bg.ac.rs, kljajiczeljko@yahoo.com, milojko.lazic@rgf.bg.ac.rs 2 University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Department of Ecology and Technoeconomics, Karnegijeva 4, Belgrade, Serbia, e-mail: spopovic.bio@gmail.com 3 University of Belgrade, Faculty of Biology, Studentski trg 16, e-mail: gsubak@bio.bg.ac.rs * Corresponding Author Received/Prejeto: 24.04.2017 COBISS: 1.01 ACTA CARSOLOGICA 46/2−3, 295–316, POSTOJNA 2017 Abstract UDC 553.7:579(497.11) Vladimir Šaraba, Slađana Popović, Olivera Krunić, Gordana Subakov Simić, Željko Kljajić, Milojko Lazić: Mineral waters of Serbia and development of phototrophic microbial commu- nities near points of emergence and on wellheads Phototropic microorganisms in thermal mats had not attracted enough attention in Serbia in the past. The research present- ed in this paper is the first to address biofilms at selected oc- currences of mineral water in Serbia (Bogatić, Metković and Belotić in the Mačva District as well as Radaljska Banja, Luk- ovska Banja and Vranjska Banja) (in Serbian, banja denotes a spa town). The study period is from 2014 to 2016. The temper- atures of the studied occurrences are in the 30–93.9 °C range and the chemical composition corresponds to the HCO3 − – Na++K+ and SO4 2−, HCO3 − – Na++K+ types of mineral water. One of the objectives was to examine the condition of wellheads (incrustation, biofouling and corrosion), focusing on the pres- ence of phototrophic microorganisms whose metabolites may lead to changes in mineral water quality, yield and chemistry. Analysis by light and scanning electron microscopy provides insight into the diversity of phototrophic microorganisms that populate biofilms near the points of emergence and on well- head. The research reveals the presence of representatives of three groups of phototrophic microorganisms: Cyanobacteria, Chlorophyta and Bacillariophyta. Cyanobacteria were the most numerous, dominated by the order Oscillatoriales with ten re- corded genera, of which Leptolynbgya and Phormidium feature the largest numbers of species. The order Chroococcales are the second most numerous, with seven recorded genera, of which gloeocapsa and Synechocystisare the most noteworthy. The largest numbers of representative taxa were noted at Radaljska Banja and Vranjska Banja, where mineral waters originate from Izvleček UDK 553.7:579(497.11) Vladimir Šaraba, Slađana Popović, Olivera Krunić, Gordana Subakov Simić, Željko Kljajić, Milojko Lazić: Mineralne vode v Srbiji in razvoj fototrofnih mikrobnih združb na mestih izvi- rov in na ustjih vrtin Fototrofni mikroorganizmi v oblogah termalnih izvirov v Srbiji v preteklosti niso bili deležni posebne pozornosti. V članku predstavljena raziskava je prva, ki obravnava biofilme na izbranih mestih izvirov mineralnih vod v Srbiji (Bogatić, Metković in Belotić v Mačvi ter Radaljska Banja, Lukovska Banja in Vranjska Banja) (v srbskem jeziku je banja izraz za toplice). Preučevanje je potekalo med letoma 2014 in 2016. Temperature obravnavanih voda se gibljejo med 30 in 93,9 °C, glede na kemijsko sestavo pa jih uvrščamo v HCO3 − – Na++ K+ in SO4 2−, HCO3 − – Na++K+ tipe mineralne vode. Eden od ciljev raziskave je bil proučevati razmere na ustjih vrtin (inkru stacija, obrast in korozija) in se še posebej posvetiti navzočnosti fo- totrofnih mikroorganizmov, katerih metaboliti lahko spre- menijo kakovost, izdatnost in kemizem mineralnih vod. Z analizo slik s svetlobnim in vrstičnim elektronskim mik- roskopom lahko podrobneje spoznamo raznolikost fototrof- nih organizmov, ki sestavljajo biofilme na mestih izvirov in na ustjih vrtin. Raziskava je pokazala obstoj predstavnikov treh skupin fototrofnih mikroorganizmov: cianobakterij ter predstavnikov debel Chlorophyta in Bacillariophyta. Cianoba- kterije so bile najbolj številčne, prevladovali so osebki reda Oscillatoriales z desetimi ugotovljenimi rodovi, od katerih je bilo največje število vrst iz rodov Leptolynbgya in Phormidium. Red Chroococcales je bil s sedmimi zabeleženimi rodovi drugi najbolj številčen, prevladovala sta gloeocapsa in Synechocystis. Največreprezentativnih taksonov je bilo v Radaljski Banji in Vranjski Banji, kjer mineralne vode izvirajo iz litostratigrafskih ACTA CARSOLOGICA 46/2–3 – 2017296 VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć INTRODUCTION Different geologic, chemical and other conditions make each occurrence of groundwater a unique ecosystem, which is more or less complex and where microorgan- isms are acclimated to the particular setting. They persist owing to special physiological and ecological adaptations and a variety of distinct properties (Griebler & Lueders 2009). The presence of microorganisms in groundwater and near the place of emergence is constrained by nu- merous factors. The impact of the pH level, ionic activity and temperature is unavoidable (Donlan 2002; Li & Lo- gan 2004). The microbial count also depends, inter alia, on the concentrations of dissolved organic and inorganic carbon, as well as other nutrients in the groundwater (Lehman 2001). In most cases, microorganisms survive as long as enough resources are available for growth and reproduction (Lawrence & Caldwell 1987; Szewzyk & Schink 1988). In groundwater and near the point of emergence, microorganisms tend to build biofilms attached to the rock matrix (Hazen et al. 1991; Griebler et al. 2001). Biofilms are considered to be the earliest ecosystems on the planet Earth (Tice & Lowe 2004; Noffke et al. 2006); they have been around for more than three billion years (Hoffman et al. 1999; Schopf 2006). Over time, they have altered the oxidation-reduction conditions on Earth (Des Marais 1995; Farmer 2000). It is believed that biofilms spearheaded the process of photosynthesis (Des Marais 2000; Kasting & Howard 2006). Biofilm ecosystems are extremely efficient in the circulation of elements in na- ture (Visscher & Stolz 2005). The photosynthetic capabil- ity, light utilization efficiency, and rate of procreation of phototrophic biofilm algae constitute some of the most powerful mechanisms that covert sunlight into organic matter. Owing to these characteristics, microalgae and cyanobacteria are extremely important in biotechnology. Biofilms are also an ideal substrate for astrobiological re- search (Toporski et al. 2003), given that the study of life in such extreme environments parallels that of potential life on other planets (Pedersen 2000). At all the studied mineral water sites in Serbia, the production wells have aged. Well ageing has an adverse effect on the structural and/or hydraulic characteristics of the well screen and the aquifer matrix around the screen. It comprises all process related to the degradation of materials due to corrosion, as well as mechanical and chemical clogging of screen slots, the gravel pack and the zone of the aquifer adjacent to the screen. In nearly all cases, well ageing leads to the deterioration of hydraulic characteristics (Driscoll 1986; Houben & Treskatis 2007). During the course of operation, wells tend to exhibit five major problems (Driscoll 1986): (i) declining capacity, due to chemical incrustation or biofouling in and around the screen, (ii) mechanical clogging – sanding caused by corrosion of the screen or well tube, (iii) electrolytic cor- rosion – usually where the pH level of the groundwater is low and CO2 concentrations high, (iv) biofouling, and (v) chemical incrustation. The main types of incrusta- tion in Serbia are related to the precipitation of calcium and magnesium carbonates and sulfates and iron and manganese compounds, primarily their hydroxides and hydrated oxides, but also those that are a result of me- chanical and chemical activity of various microorgan- isms. These are also the most frequent forms of incrusta- tion in worldwide hydrogeological practice, as reported by: Driscoll 1986; Houben & Treskatis 2007. Encrusta- lithostratigraphic units of igneous origin, followed by mineral water occurrences in karst aquifers (Bogatić and Belotić) and an intergranular aquifer (Metković). The smallest number of identified phototrophic microorganisms is noted at Lukovska Banja, where the origin is associated with a fractured aquifer formed in sedimentary and metamorphic rocks, with occa- sional diabase interbeds. Although many representative taxa are found on only one of the studied localities, such as Syn- echococcus bigranulatus and Pseudanabaena thermalis, those of the genera Leptolyngbya, Phormidium and Cosmarium laeve, as well as genera of the phylum Bacillariophyta, occur at several sampling sites. Principal component analysis (PCA) was used to show the relationship between documented cyanobacterial and algal taxa and environmental parameters. Key words: Serbia, mineral waters, phototrophic microorgan- isms, biofilm. enot vulkanskega izvora, sledijo mineralne vode iz kraškega (Bogatić in Belotić) in medzrnskega vodonosnika (Metković). Najmanjfototrofnih organizmov je bilo v vzorcu iz Lukovske Banje, kjer vode izvirajo iz razpoklinskih vodonosnikov v sedi- mentnih in metamorfnih kamninah z vmesnimi lečami dia- baza. čeprav soštevilne reprezentativne taksone našli le na eni od proučevanih lokacij, kot na primer Synechococcus bigranu- latus in Pseudanabaena thermalis, se tisti iz rodu Leptolyngbya, Phormidium in Cosmarium laeve in predstavniki več rodov, ki pripadajo deblu Bacillariophyta, pojavljajo na več vzorčnih mestih. Za ugotavljanje odvisnosti med taksoni cianobakterij in alg ter okoljskimi parametri so uporabili analizo glavnih komponent. Ključne besede: Srbija, mineralne vode, fototrofni mikroorga- nizmi, biofilm. ACTA CARSOLOGICA 46/2–3 – 2017 297 MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... tions of biological origin are comprised of microbial communities and mineral substances precipitated either from the groundwater or by the microorganisms them- selves. The deposits are initially small, dominated by the biological component. However, over time, mineral sub- stances acquire an increasing share and ultimately make up most of the mass. The end products tend to constitute large obstacles for groundwater flow. It is estimated that 80 % of encrustations are of biological origin (Schnie- ders 2003). The composition of biofilms also depends on the physical and chemical characteristics of the substrate on which they develop, the bacterial species that colonize them, and the type of material (LeChevallier et al. 1996). Different pipes in contact with water release various sub- stances that are a source of nutrients for the microorgan- isms which, after they establish a community, tend to spread rather quickly. Pipes that contain polyamide and silicon can cause bacterial proliferation, as can materi- als that include bitumen, epoxy resin or tar-epoxy resin (Schoenen 1986). The interactions between microorgan- isms and materials need to be seriously considered dur- ing in the design stage of groundwater extraction and transport facilities (Beech & Sunner 2006). There is another important component of the study of phototrophic microbial mats near the points of emer- gence of mineral water and on wellheads in Serbia, given that extremophiles able to produce various secondary metabolites and other thermostable compounds have been detected. Cyanobacteria and algae often adapted to very harsh conditions. The benefits of algae have been known since ancient times, owing to their anti-inflam- matory and anti-microbial properties. The importance of algae to the human population is multi-faceted: fixing of atmospheric nitrogen, creation of toxins, and primary production – organic substances and products of pho- tosynthesis, as well as because of secondary metabolites, economic significance and bioindication (Cvijan 2013; Šaraba & Krunić 2017). The development of phototrophic microbial com- munities near the points of emergence of mineral water and on wellheads in Serbia had not previously been re- searched. One of the objectives of the present study was to evaluate the status of wellheads (incrustation, bio- fouling and corrosion that may lead to changes in water quality and yield), and to conduct chemical analyses of the mineral waters. Another objective was to assess cy- anobacterial and algal diversity in thermal mats near the point of emergence and on wellheads. Also, an attempt was made to establish a correlation between document- ed phototrophic taxa and measured environmental pa- rameters (physicochemical water characteristics) using multivariate analyses, as well as between the geologic en- vironment in which the mineral waters originate (karst, fractured and intergranular aquifers) and the microbial community on the ground surface, given that groundwa- ter acquires the properties of the rocks through which it flows. This, inter alia, has an effect on phototrophic microorganisms, along with the microclimate and other environmental factors. STUDy AREAS The studied occurrences of mineral waters are found in the Inner Dinarides of western Serbia – Bogatić, Metković and Belotić in the Mačva District and Radaljs- ka Banja; in the Šumadija-Kopaonik-Kosovo province – Lukovska Banja, and the Serbian Crystalline Core – Vranjska Banja (Filipović et al. 2005), (Fig. 1). The zoning is based on tectonics, lithofacies, geomorphol- ogy, hydrogeology, and physical, geographic and other characteristics used to identify the geologic provinces of Serbia (Krunić 2012). The studied occurrences of mineral water in the Mačva District (Bogatić, Metković and Belotić) are formed within the basin structure, in Triassic limestone, Triassic pyritized dolomite and Eocene sandstone aqui- fers, underlain by plutonites which, among other things, cause elevated temperatures of the mineral waters. Karst and intergranular aquifers are tapped on those loca- tions. The fractured aquifer at Radaljska Banja is found in compact and fractured polymineralic igneous rocks (granodiorites), whereas that at Lukovska Banja occurs in Cretaceous sandy, oolitic, stratified, banked and mas- sive limestones, with serpentinite, diabase-chert, sand- stone, marl, conglomerate and mudstone components. At Vranjska Banja, mineral water is captured from a matrix of metamorphic and igneous rocks: micaschists, leptino- lites, gneisses, gneiss-granites and a volcanic sedimenta- ry complex, represented by Neogene volcanic dust, sand and ash (Filipović et al. 2005). The studied mineral wa- ters are tapped from different geologic media, including karst (Bogatić, Belotić and Lukovska Banja), fractured (Radaljska Banja and Vranjska Banja) and intergranular (Metković) aquifers, whose hydrodynamic character- istics differ. Some of the aquifers are unconfined, while others are confined. ACTA CARSOLOGICA 46/2–3 – 2017298 From a tectonic perspective, the Mačva District is a wing of the large Sava trench, between Mt. Cer and Mt. Fruška Gora, where a regional fault trending west-east runs along the valley of the Sava River. The trench was formed in several stages, ending with the Pleistocene. Radaljska Banja belongs to the large Drina anticline, cross-crossed by primary fault structures and a series of lower-order faults, perpendicular to each other. They play a significant role in the formation of mineral water on this locality. The boundaries are clearly dominated by a fault zone, intersected by subsequent lateral faulting. Vranjska Banja is part of the Vranje trench and the oc- currences of mineral water are associated with a folding zone, where in relative terms the block south and south- fig. 1: Schematic representation of the geographic and geotectonic positions and geologic microlocation frameworks of the studied oc- currences of mineral waters in Serbia. Legend: I – Dacian Basin; II – Carpathian-Balkanide province; III – Serbian Crystalline Core; IV – šumadija-kopaonik-kosovo prov- ince; V – Inner Dinarides of western Serbia; VI – Pannonian Plain; a) geological section at Bogatić (BB-1), Metković (BMe-1)and Belotić (BBe-1) –Mačva region; b) geological section at Radaljska Banja (RB-3/90); c) geological section at Lukovska Banja (LB-4 and LB-5); d) geological section at Vranjska Banja (Vg-2 and Vg-3); 1 – Neogene sediments: coarse gravel, marly clay, sand, clay, sandstone, conglomerate; 2 – Igneous sedimentary complex; 3 – tuff; 4 – Eocene sandstone; 5 – granite; 6 – Limestone, marly lime- stone, slate and sandstone; 7 – Limestone, marl and sandstone; 8 – Limestone, coarse banked sandstone, marl, conglomerate, siltstone; 9 – Limestone, siltstone, sandstone, marl; 10 – Limestone, sandstone, conglomerate, siltstone; 11 – Diabase-chert formation: sandstone, limestone, chert and slate; 12 – triassic limestone; 13 – granodiorites and granodiorite-porphyritic rocks; 14 – gneiss, micaschist and leptinolites; 15 – Serpentinite; 16 – geological boundary; 17 – fault; 18 – Well. VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV-SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 299 MATERIALS AND METHODS Detailed geological investigations began with a review of available documentation pertaining to several decades of regional hydrogeological exploration, including informa- tion about previous physical and chemical testing of min- eral water and the geologic framework within the zones of emergence of mineral water (Fig. 1). The review was followed by hydrogeological reconnaissance of the study areas. The occurrences and wells were identified and then sampling conducted accordingly. Given the topic and objectives of the research, the materials and methods related to geologic exploration included sampling of: (i) mineral waters, along with physical and chemical analy- ses, and (ii) encrustations. The biological stage of the research focused on proper sampling of the phototropic biofilms, which had been formed over time on the wellhead and near the point of emergence of mineral water, followed by light and scanning electron microscopy and identification of microorganisms. These steps of the research allowed for symbiosis of biological and geologic facts. SITE DESCRIPTION AND SAMPLING The present paper is an outcome of previous multiyear regional hydrogeological exploration of mineral waters in Serbia as well as specially designed investigations con- ducted from 2014 to 2016. Mineral water is groundwater whose temperature is above 20 °C, total dissolved solids (TDS) greater than 1 g/l, and/or that which contains elevated concentra- tions of certain chemical components (macro and mi- cro), features elevated radioactivity, and includes free and dissolved gases, biologically active components, and the like, whose properties constitute a special class of groundwater (Krunić 2012). The mineral water occurrences in Serbia were selec- ted either because of their significance in terms of use (Radaljska Banja, Lukovska Banja and Vranjska Banja – balneotourism) or their potential and physical proper- ties such as in the Mačva District (Bogatić, Metković and Belotić).The selection was also influenced by practical reasons: for example Bogatić (need to rehabilitate well BB-1), and the fact that one of the objectives was to in- vestigate areas of different structural geology, lithofacies, hydrogeology and other characteristics, as well as mine- ral water occurrences of different temperatures. In the past, mineral water occurrences had been addressed from the viewpoint of geologic framework, characteristic rock complexes in which they are found, basic ionic and gas compositions, specific components, likely primary origin, fundamental and specific proces- ses that lead to the formation of the chemical compositi- on, regime, reserves, and potential for multi-purpose use (Krunić et al. 2016). The development of phototrophic organisms in biofilms near the points of emergence of mineral water and on wellheads has not previously been studied in Serbia. It should be noted that past hydrogeological explo- ration had often been affected by microbial activity at the wells and near the points of emergence of ground- water, but no appropriate biological research was un- dertaken. One of the significant aspects of the research con- ducted from 2014 to 2016 was the identification of pho- totrophic microorganisms on wellheads, which, among other things, led in well ageing. west of Vranjska Banja rises and the central part sinks. The tectonic complexity of Lukovska Banja is attributed to the fact that mineral water occurs at the very contact between large regional tectonic units of the western “Ex- ternal Vardar Sub-zone” and the eastern “Central Vardar Sub-zone”. In general, the terrain was created in several stages, which led to considerable vertical and horizon- tal displacements. The first resulted in uplifting of the central part of the terrain and deposition of Cretaceous sediments along the western and eastern edges. In the Upper Cretaceous, there was intensive tectonic move- ment that created the Central Vardar Sub-zone”. Uplift- ing of the Jurassic and Jurassic-Cretaceous complex and the central and eastern tectonic units continued. Strong post-Senonian tectonic movements resulted in consider- able horizontal and vertical displacement of the blocks, particularly in the central zone of the studied site, as well as folding of the Cretaceous complex in the eastern and western parts, which drove the formation and emergence of mineral water (Filipović et al. 2005; Krunić 2012). All of the above have impeded the determination of hydro- geologic structures and mineral water flow rates, which have not been accurately established to date. Vertical sections through the geologic framework of the areas where mineral waters emerge and are tapped are shown in Figure 1; the relevant zones are identified by blue dots in the lower left corner. MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017300 The mineral water sampling points were near mi- neral water emergences and on wellheads (Fig. 2). The points of emergence (or discharge) of mineral water are places where mineral water reaches the ground surface via a free-flowing well or well whose column leaks due to ageing or drained naturally (Dragišić & Polomčić 2009), whereas the wellhead is the place on the ground surface where the extraction well begins (Lazić 2003). The characteristics of the studied mineral waters are shown in Tab. 1. Mineral waters were sampled for chemical and physicochemical analyses at eight locations (Bogatić – well BB-1; Metković – well BMe-1; Belotić – well BBe-1; Radaljska Banja – well RB 3/90; Lukovska Banja – wells LB-4 and LB-5; and Vranjska Banja – wells VG-2 and VG-3). Contrary to sampling of mineral waters for chemical analyses, the discharge zones of the wells at Lu- kovska Banja (wells LB-4 and LB-5) and Vranjska Banja (wells VG-2 and VG-3) were close together and covered by the same type of biofilm, such that sampling for mi- crobiological analyses was undertaken simultaneously at both wells in Lukovska Banja and Vranjska Banja. A total of six discharge zones were sampled for microbiological analyses. Sampling of mineral waters and biofilms was con- ducted over a two-year period, from 2014 to 2016. At Bogatić, Metković and Belotić, samples were collected in mid-December 2014 – when autumn ended and winter began, and on the other locations (Radaljska Banja, Luk- ovska Banja and Vranjska Banja) in April and May 2015. PHySICAL AND CHEMICAL ANALySES OF MINERAL WATERS Samples of mineral water for physical and chemical analy- ses were collected in plastic bottles. The bottles were ster- ile and the caps complied with international standards ISO 9001, ISO 14001 and OHSAS 18001. Prior to use, the bottles were rinsed and cleaned with a 1 % HCl solution, then flushed with distilled water. Upon sampling of min- eral water, the bottles were capped to prevent leakage and then sealed with a nut. The bottles were labeled, indicat- ing serial numbers and other important information, like the name of the sampling site, water temperature (T), pH, and total dissolved solids (TDS). Mineral water samples for physicochemical analyses and biofilm samples for microbiological analyses were collected at the same time. The temperature (T), pH and conductivity (EC) of the mineral waters were determined in situ (Tab. 2). CyberScan CON 510 pH meter was used to deter- mine pH levels and temperatures. This instrument could also be used to measure total dissolved solids (TDS). Conductometer HI98192 was used to measure con- ductivity. This instrument is equipped with a four-ring HI763133 conductivity electrode. The other parameters were analyzed in the labo- ratory for physical and chemical analyses of Belgrade’s Public Health Institute (TDS, Na++ K+, Ca2+, Mg2+, Cl−, SO4 2−, NO2 −, NO3 −, NH4 +) (Tab. 2). MICROSCOPIC EXAMINATIONS The microbial mat samples were analyzed using light and electron microscopy (SEM-EDS analysis) at the laborato- ries of the University of Belgrade/Faculty of Biology and Faculty of Mining and Geology. Algological analyses. Mat samples were collected near the points of emergence of mineral water and at wellheads in Bogatić, Metković, Belotić, Radaljska Ban- ja, Lukovska Banja and Vranjska Banja (Fig. 2). Sam- pling sites with notable highly-developed and variously colored biofilm were chosen for analysis. The samples were collected using a sterilized scalpel, placed in ster- ile plastic bottles and fixed with a 1.5 % formaldehyde solution. The samples were analyzed using a light micro- scope (Zeiss Axio Imager-M1 with AxioVision 4.8 soft- tab. 1: Characteristics of studied occurrences of mineral waters (Martinović & Milivojević 1998; krunić 1999; filipović 2003; filipović et al. 2005; krunić 2012; Martinović et al. 2012). Locality Well Depth of well (m) Lithology Yield (l/s) Type of mineral water Bogatić BB-1 470 Triassic limestone 37.5 HCO3−Na+K Metković BMe-1 370 Neogene sandstone from quartz minerals with lime binder 15 HCO3−Na+K Belotić BBe-1 627 Triassic dolomite, pyritized 10 HCO3−Na+K Radaljska Banja RB-3/90 366 Compact and fractured granodiorite 3 HCO3−Na+K Lukovska Banja LB-4 401 Cretaceous limestone 4.7 HCO3−Na+K LB-5 870 Cretaceous limestone 4.6 HCO3−Na+K Vranjska Banja VG-2 1064 Petrified granodiorite and gneiss-granite, amphibole-biotite schist 26 SO4, HCO3−Na+K VG-3 1470 Petrified quartz-diorite, grandiorite, with schist intercalations 21 SO4, HCO3−Na+K VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV-SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 301 successful. It is well-known that cyanobacteria from ex- treme environments, especially coccoid taxa, are gener- ally hard to cultivate, since the imitation of their original habitats is usually an obstacle. For that reason, our study involved only one-time screening of cyanobacterial and algal taxa. ware) and the cyanobacteria and algae were identified according to: Krieger & Gerloff 1965; Komárek & Fott 1983; Komárek & Anagnostidis 1998, 2005; Komárek 2013; Komárek et al. 2014; Lange-Bertalot et al. 2011; Hofmann et al. 2013. Despite using a new technique, the cultivation of these thermal microorganisms was not fig. 2: Biofilm sampling sites for microbiological analyses of mineral waters in Serbia (Photo: M. Lazić, 2014−2016). Legend: a) Bogatić; b) Metković; c) Belotić; d) Lukovska Banja; e) Radaljska Banja; f) Vranjska Banja. MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017302 SEM-EDS analyses. These analyses were conducted using an electron microscope. Phase compositions of powdered black crust and powdered dry residue were determined by X-ray powder diffraction (XRPD). XRPD was performed on a Philips PW1710 diffractometer. The diffraction patterns were obtained from 5 to 70° 2θ, with a step scan of 0.02° 2θ and 0.5 s of counting time at each step using CuK α radiation. The morphology and chemi- cal composition of the mineral phases present in the dry residue of water were identified using a JEOL JSM-6610- LV Scanning Electron Microscope connected to an X- Max Energy Dispersive Spectrometer. The samples were coated with gold using a BALTECSCD-005 SPUTTER coating device and recorded under high vacuum condi- tions. STATISTICAL ANALySES Principal component analysis (PCA) was performed using the program CANOCO for Windows, Version 5.0 (Ter Braak & Šmilauer 2012). PCA was applied to examine the relationship between documented cyano- bacterial and algal taxa and measured environmental variables. For project data, the presence/absence of all recorded taxa was used as a measure. Then, each taxon was assigned to a taxonomic group (Chroococcales, Os- cillatoriales and Nostocales (the three corresponding to Cyanobacteria), Chlorophyta and Bacillariophyta). In further analysis, these formed groups were used instead of individual taxa. Measured environmental parameters (physical and chemical water parameters), as well as lo- cations, were used as supplementary variables. PCA with the option “center and standardize” was applied. RESULTS AND DISCUSSION The results of the physical and chemical analyses (Tab. 2) indicated that the types of mineral water were HCO3 − – Na++K+ and SO4 2−, HCO3 − – Na++K+ (Fig. 3, Fig. 4). The concentrations of HCO3 – ranged from 80 mg/l (well RB-3/90 at Radaljska Banja) to 5980 mg/l (well VG-3 at Vranjska Banja). Sulfate ions (SO4 2−) were detected in con- centrations from 2.5 mg/l (well BMe-1 at Metković) to 548.7 mg/l (well VG-2 at Vranjska Banja). Sodium (Na+) was measured from 50 mg/l (well RB-3/90 at Radaljska Banja) to 358.0 mg/l (well VG-2 at Vranjska Banja). The lowest temperature was recorded in well RB- 3/90 at Radaljska Banja – 30 °C, and the highest in well VG-2 at Vranjska Banja – 93.9 °C. The pH level varied from 6.6 in well LB-5 at Lukovska Banja to 9.3 in well RB- 3/90 at Radaljska Banja. A noteworthy finding was TDS whose values varied over a broad range, from 175 mg/l in well RB-3/90 at Radaljska Banja to 1267 mg/l in well LB-4 at Lukovska Banja, as well as EC from 230 µS/cm at well RB-3/90 in Radaljska Banja to 1588 µS/cm at well LB-5 in Lukovska Banja. Gas analyses included carbon dioxide (CO2), oxy- gen (O2) and hydrogen sulfide (H2S). There was also a notable presence of nitrates (NO3 −), measuring 5.3 mg/l at well VG-3 in Vranjska Banja, and of ammonium ions (NH4 +) – 4.92 mg/l at well BMe-1 in Metković. Conductivity, as an electrical property of the stud- ied mineral waters, directly correlates with the ion con- tent and water temperature. The correlation between these physicochemical parameters is illustrated in Fig. 5. The identified cyanobacterial and algal taxa from mat samples collected at the points of emergence of min- eral waters are listed in Tab. 3, and some representatives are shown in Figs. 6 and 7. The analyzed microbial mats near the points of emergence of mineral water and on wellheads in Bogatić, Metković, Belotić, Lukovska Banja, Radaljska Banja and Vranjska Banja (Tab. 3) revealed the presence of cyanobacteria and algae from two groups: Chloro- phyta and Bacillariophyta. Taking into account the number of taxa, cyanobacteria were dominant. Many taxa were found at only one sampling site, like Chroo- coccus thermalis (Fig. 7b) in Vranjska Banja or Spiruli- na major (Fig. 7e) in Lukovska Banja. The majority of the taxa represented filamentous forms from the Oscil- latoriales group (ten taxa), of which Leptolynbgya and Phormidium had the highest numbers of recorded spe- cies. Seven genera were identified in the Chroococcales group and the most frequent were gloeocapsa and Syn- echocystis. Heterocytous Cyanobacteria, which belong to the Nostocales group, had only one representative of the genus Calothrix. The largest number of cyanobacte- rial taxa was found in the mat samples from Lukovska Banja and Vranjska Banja. The group Oscillatoriales was dominant in Radaljska Banja, Lukovska Banja and Belotić, while Chroococcales prevailed in Vranjska Banja. The diversity was also high in Belotić, where het- erocystous cyanobacteria were also recorded. However, some taxa were detected at more than one site, such as Synechococcus bigranulatus, Pseudanabaena thermalis, representatives of the genera Leptolyngbya and Phor- midium, Cosmarium leave, as well as members of the Bacillariophyta division. VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 303 The largest number of recorded taxa were found in fractured aquifers within rocks of igneous origin (Radaljska Banja and Vranjska Banja), followed by karst aquifers in limestones and dolomites (Bogatić and Belotić), and the intergranular aquifer at Metković. The mineral water from the fractured aquifer within rocks of sedimentary and metamorphic origin, with diabase in- terbeds (Lukovska Banja), featured the lowest number of phototrophic microbial taxa. The wellhead material was also examined in the geological research phase. Steel wellheads at the studied sites favored the development of biofilm. This has been corroborated by other researchers, who report that bio- films develop more rapidly on steel than polyvinyl chlo- ride (PVC) (Norton & LeChevallier 2000; Beech & Sun- ner 2006). The biodiversity at wellheads was assessed at Bogatić, Metković, Belotić and Vranjska Banja, as part of the biological research phase. tab. 2: Results of physical and chemical analyses of studied occurrences of mineral water (Public health Institute of Belgrade, 2015). Parameter BB-1 BMe-1 BBe-1 RB-3/90 LB-4 LB-5 VG-2 VG-3 T (°C) 65.9 59.8 35.0 30.0 68.1 62 93.9 91.2 TDS (mg/l) 600 1082 850 175 1267 1058 1213 1145 pH 6.9 7.0 7.2 9.3 7.5 6.6 7.9 8.0 T (NTU) / / / 1 / 14.9 0.6 0.8 KMnO4 demand (mg/l) 6.9 9.2 8.7 1.7 2.2 3.2 5.5 4.2 Color (PT-Co scale) / / / 0 / 25 <5 <5 EC (µS/cm) 800 1443 1133 230 1513 1588 1584 1707 MACROCOMPONENTS(mg/l) Na+ 144.0 251.6 158.0 50.0 309.6 166 358.0 346.4 K+ 13.0 19.8 13.9 0.7 23.6 18.6 19.4 18.4 Ca2+ 33.5 34.7 47.7 2.2 41.6 83.2 12.8 16.0 Mg2+ 6.6 7.8 10.6 0.07 17.1 32 <1.0 <1.0 HCO3 − 361.6 640.0 470.0 80 961.0 961.0 589.0 5980.0 SO4 2− 7.5 2.5 3.0 12 91.3 50.3 548.7 487.0 Cl− 103.4 112.7 106.8 5 82.0 46.0 64.0 60.0 MICROCOMPONENTS (mg/l) Pb <0.003 <0.003 <0.003 <0.001 / 0.04 <0.01 <0.01 Ni / / / / / 0.11 <0.01 <0.01 Ag / / / / / <0.013 / / Cd / / / <0.001 / / <0.02 <0.02 Hg / / / <0.0002 / / <0.001 <0.001 As 0.002 0.002 0.002 <0.001 / 0.689 <0.01 <0.01 Zn 0.002 0.002 0.004 0.002 / <0.014 <0.02 <0.02 Free CO2 50.5 96.8 13.02 <0.5 / / 184.6 194.2 Dissolved O2 0.5 2.5 0.5 1.6 / / 4.5 3.7 Free H2S 2.5 0.8 0.6 2.1 / <0.06 0.008 0.006 NO3 − 0.3 <0.02 <0.2 <0.2 0.7 0.5 2.3 5.3 NO2 − 0.002 0.003 0.003 <0.005 0.005 <0.005 <0.02 <0.02 NH4 + 1.80 4.92 2.46 <0.05 1.05 0.75 0.31 0.32 Legend: BB-1 – well in Bogatić; BMe-1 – well in Metković; BBe-1 – well in Belotić; RB-3/90 – well in Radaljska Banja; LB-4 – well in Lukovska Banja; LB-5 – well in Lukovska Banja; Vg-2 – well in Vranjska Banja;Vg-3 – well in Vranjska Banja; / –parameter not determined. MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017304 fig. 3: Piper diagram of the chemical composition of mineral waters in Ser- bia. fig. 4: Stiff diagram of the chemi- cal composition of mineral waters: Bogatić (BB-1), Metković (BMe-1), Belotić (BBe-1), Radaljska Banja (RB- 3/90), Lukovska Banja (LB-4, LB-5) and Vranjska Banja (Vg-2, Vg-3). VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 305 Along with other environmental factors, microcli- mate conditions affect the development of thermal bio- films. The climate in the Mačva District (Bogatić, Metković and Belotić) and Radaljska Banja is moderately continental, while that at Lukovska Banja and Vranjska Banja is moderately continental but modified by the in- fluence of mountainous climate (Filipović et al. 2005). The perennial average precipitation (Pav) and air temperature (T) in the study areas were: Bogatić, tab. 3: Identified cyanobacterial and algal taxa. Identified phototrophic microorganisms 1 2 3 4 5 6 Cyanobacteria Chroococcales Aphanocapsa cf thermalis (Brügger) + Aphanothece sp. (C. Nägeli) + Chroococcus thermalis (Meneghini & Nägeli) + Cyanosarcina thermalis (Hindák & Kovácik) + Gloeocapsa arenaria (Hassall & Rabenhorst) + + + + Gloeocapsa gelatinosa (Kützing) + + + Synechococcus bigranulatus (Skuja) + + Synechocystis thermalis (J. J. Copeland) + Synechocystis sp. (C. Sauvageau) + Oscillatoriales Borzia trilocularis (Cohn ex Gomont) + Geitlerinema amphibium (C. Agardh ex Gomont & Anagnostidis) + Geitlerinema thermale (Anagnostidis) + Jaaginema angustissimum (West, G. S. West, Anagnostidis & Komárek) + Jaaginema geminatum (Schwabe ex Gomont, Anagnostidis &Komárek) + Komvophoron jovis (J. J. Copeland, Anagnostidis & Komárek) + Leptolynggbya angustissima (West & G. S. West, Anagnostidis & Komárek) + Leptolyngbya foveolarum (Gomont, Anagnostidis & Komárek) + + Leptolyngbya granulifera (J. J. Copeland & Anagnostidis) + + Leptolyngbya laminose (Gomont ex Gomont, Anagnostidis & Komárek) + + + Leptolyngbya orientalis (G. S. West, Anagnostidis & Komárek) + + Leptolyngbya thermalis (Anagnostidis) + Leptolyngbya spp. (Anagnostidis & Komárek) + + + Microcoleus chthonoplastes (Thuret ex Gomont) + Oscillatoria princeps (Vaucher ex Gomont) + + Phormidium spp. (Kützing ex Gomont) + + + + Phormidium grunowianum (Gomont, Anagnostidis & Komárek) + + Phormidium jasorvense (Vouk, Anagnostidis & Komárek) + Phormidium terebriforme (C. Agardh ex Gomont, Anagnostidis & Komárek) + Phormidium tergestinum (Rabenhorst ex Gomont) Anagnostidis & Komárek) + Pseudanabaena thermalis (Anagnostidis) + + + + Spirulina major (Kützing ex Gomont) + Nostocales Calotxrix thermalis (Hasngirg ex Bornet & Flahault) + Chlorophyta Cosmarium laeve (Rabenhorst) + + + Bacillariophyta Epithemia sp. (Kützing) + Navicula spp. (Bory) + + + + + Nitzschia spp. (Hassall) + + Legend: 1 – well BB-1 (Bogatić); 2 – well BMe-1 (Metković); 3 – well BBe-1(Belotić); 4 – well RB-3/90 (Radaljska Banja); 5 – wells LB-4 and LB-5 (Lukovska Banja); 6 – wells Vg-2 and Vg-3 (Vranjska Banja). MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017306 Metković and Belotić – Pav = 676 mm, T = 11.2 °C; Radaljska Banja – Pav = 1136 mm, T = 11.0 °C; Lukovska Banja – Pav = 607.9 – 1570 mm, T = 10.4 °C; and Vranj- ska Banja – Pav = 568.9 mm, T = 11.3 °C (Martinović & Milovojević 1998; Krunić 1999; Filipović 2003; Filipović et al. 2005; Martinović et al. 2012). Biofilms at Bogatić, Metković and Belotić were sampled in December 2014, when autumn turned into winter. December 2014 was characterized by a cold au- tumn and severe winter. At the time of sampling, there was a snow cover around some of the wells. During the spring months of 2015, when sampling was undertaken at Radaljska Banja, Lukovska Banja and Vranjska Banja, there was a lot of rain and humidity was high. PCA (Fig. 8) was used to illustrate the relation- ship between documented cyanobacterial and algal taxa and measured environmental variables. All measured environmental variables (physical and chemical water parameters, macronutrients and micronutrients) were submitted to PCA, but only those showing high correla- tion coefficients with the first two PCA axes were shown in the ordination diagram. It is apparent that Ca2+ and Mg2+ correlate with the positive part of the first PCA axis (r = 0.8095 and r = 0.8418, respectively). Many other variables are correlated with the negative part of the first PCA axis: NO3 − (r = −0.5273), NO2 − (r = −0.6342), SO4 2− (r = −0.5476), HCO3 − (r = −0.4916), pH (r = −0.4297) and T (r = −0.3404). H2S correlated with the negative part of the second PCA axis (r = −0.5662). Chroococ- cales that prevailed in Vranjska Banja showed a positive correlation with increasing T, indicating that coccoid cyanobacterial taxa are the most adaptive to high tem- peratures. They also exhibited a positive correlation with pH, nitrogen compounds and other parameters that neg- atively correlated with the first PCA axis. On the other hand, Nostocales did not correlate with the measured factors (including nitrogen compounds), except for a negative correlation with H2S. The non-correlation with nitrogen compounds was expected, since Nostocales are capable of fixing nitrogen. Oscillatoriales, Chlorophyta and Bacillariophyta were negatively correlated with T. Debnath et al. (2009) also concluded that some mem- bers of the Oscillatoriales order (i.e. Phormidium sp.) are negatively correlated with T and pH. The temperature, pH value in the near-well region, physical and chemical characteristics of the mineral wa- ter, and interactions of the water with the aquifer matrix have a considerable effect on the survival and growth of microorganisms, as well as on the spatial variability of the taxa identified in the mineral water ecosystem and biofilm. Psychrophiles are microorganisms capable of growth in low-temperature environments, from −5 °C to +20 °C. They can even survive in colder temperatures. Mesophiles are microorganisms that grow in moder- ate temperatures, the optimum range being 30–45 °C. Thermophiles grow in high-temperature environments (above 45–50 °C), whereas extremophiles survive in temperatures of 40–100 °C or higher (Jemcev & Đukić 2000) (Fig. 9). One of the most important factors that influence the diversity of microorganisms in thermal mats is tem- perature. It has been reported that microbial diversity decreases with increasing temperature (Debnath et al. fig. 5: ternary diagram of tem- perature (t), conductivity (EC) and total dissolved solids (tDS) of the tested mineral water sam- ples. VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 307 2009), such that mineral waters with temperatures over 50 °C (i.e. Bogatić, Metković, and Vranjska Banja) rep- resent localities inhabited by specialized thermophilic species (Kaštovský & Komárek 2001). The majority of the identified phototrophic taxa are thermophilic spe- cies (Fig. 9), but a few can also be found in other habitats (Jemcev & Đukić 2000). All researched localities belong to the category of mineral water, with a gradation of hypo-, homeo- to hyperthermia (Fig. 9). Temperature is one of the main factors that determine the distribution and abundance of species due to its effect on enzymatic activity (Aguilera et al. 2012). The pH value also has a significant effect on the growth and survival of microorganisms in the mineral water ecosystem. Mineral waters are in the pH range from slightly acidic to alkaline (Fig. 10a). Accordingly, the identified microorganisms are considered to be neutrophils, capable of tolerating an alkaline environ- fig. 6: SEM-EDS analysis of mat samples from Bogatić, Metković, Belotić, Lukovska, Radaljska and Vranjska Banja. Legend: a, c – Colonial globular structure of framboidal pyrite; b – Nitzschia spp.; d –Pseudana- baena thermalis; e – Cyanobacte- ria covered by calcite crystals; f, g – Cyanobacterial filaments em- bedded in extracellular polymeric substance (EPS); h – Chroococcal cyanobacteria. MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017308 ment (Fig. 10b). The survival of the identified microbial communities in the pH range from 6.5 to 9.5 (Radaljska Banja) is aided by their ability to adapt to surrounding extracellular conditions. In general, the microbial cyto- plasm is characterized by a neutral reaction and a large number of important cell constituents can decompose in an alkaline medium (RNA, phospholipids) (Jemcev & Đukić, 2000). fig. 7: Cyanobacteria and algae identified by light optical mi- croscopy of biofilm sampled at Bogatić, Metković, Belotić, Lu- kovska Banja, Radaljska Banja and Vranjska Banja. Legend: a – Synechococcus bri- angulatis; b – Chroococcus ther- malis; c – Borzia trilocularis; d – Cosmarium leave; e – Spirulina major; f – geitlerinema ther- male; g – Pseudanabaena ther- malis; h – Phormidium sp.; Scale bar – 10 µm. VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 309 The survival of microorganisms in thermal biofilm depends on the chemical characteristics of the mineral water. Water chemistry is directly influenced by the geo- logic setting, given that the water acquires the properties of the rocks through which it flows, and is at the same time a consequence of subsequent interactions of water with the rock matrix in the zone of discharge. Water dis- solves solid phase particles, which enter the liquid phase where microorganisms are able to use them. At certain sites there is further interaction of mineral water with compact rock and denuded material. If the composition of the water falls below the re- quired minimum, life functions cease and the micro- organisms enter a latent (anabiotic) state and no longer reproduce (Jemcev & Đukić 2000). The mineral waters of Lukovska Banja and Vran- jska Banja are characterized by elevated concentrations of nitrogen compounds (Tab. 2): LB-4 – 0.7 mg/l NO3, LB-5 – 0.5 mg/l NO3, VG-2 – 2.3 mg/l NO3 and VG-3 – 5.3 mg/l NO3. This is one of the main nutrients for the development of algae and cyanobacteria. Non-heterocy- tous forms of cyanobacteria prefer areas that are rich in nitrogen, compared to heterocytous forms that are usu- ally found in places with lower concentrations of this nu- trient (Debnath et al. 2009). Elevated concentrations of sulfides have an adverse effect on the development of cy- anobacteria and algae, since they can inhibit photosyn- thesis (Ferrari et al. 2002). High concentrations of this ion were recorded at mineral springs in Mačva (sampling site BB-1 – 2.5 mg/l H2S) and Radaljska Banja (sampling site RB-3/90 – 2.1 mg/l H2S). Heavy metals (lead, cad- mium, mercury, nickel, silver) (Tab. 2) were generally detected in mineral waters at all the localities. The hydrogeologic aspect of the research of the growth of phototrophic microorganisms in the vicinity of the points of emergence of mineral water and on well- heads is of an applicative nature. It focuses on the role in the creation of precipitates, i.e. biofouling and incrusta- tion of well casing and wellhead. These processes, alone or in synergy, cause well ageing (Driscoll 1986; Houben & Treskatis 2007). For example, the discharge capacity of well BB-1 at Bogatić decreased from 60 l/s at a pressure of 2.9 bar in 1986 to 37 l/s at a pressure of 2.5 bar in 2014, as established by a hydrodynamic test (Fig. 11). Mineral salt and alkali precipitates are formed as a resulted of a fig. 8: PCA ordination diagram showing the relationship between cyanobacterial (Chroococcales, Oscillatoriales, Nostocales) and algal (Chlorophyta and Bacillariophyta) taxa arranged in five groups and environmental parameters (Ca2+, Mg2+, NO3 −, NO2 −, SO4 2−, hCO3 −, ph, and h2S). Red squares indicate the locality, in- cluded as a supplementary variable (BB – Bogatić, BBe – Belotić, BMe – Metković, RB – Radaljska Banja, VB – Vranjska Banja, and LB–Lukovska Banja). fig. 9: Classification of mineral waters (a) and microorganisms (b) based on temperature. Legend: BB-1– well in Bogatić; BMe-1 – well in Metković; BBe-1 – well in Belotić; RB-3/90 – well in Radaljska Banja; LB-4 – well in Lukovska Banja; LB-5 – well in Lukovska Banja; Vg-2 – well in Vranjska Banja; Vg-3 – well in Vranjska Banja. MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017310 supersaturated solution. When the product of solubil- ity of a potentially precipitating compound is exceeded, it becomes deposited (in this case calcium carbonate). When the pressure is sub-atmospheric, and particular- ly where the temperature is elevated, carbon dioxide is freed from the water. This changes the equilibrium of the solution and causes low-solubility calcium carbonate to be precipitated. The concentration of free carbon dioxide can be higher than needed to keep calcium carbonate in solution. The excess is aggressive towards limestone and will react with calcium carbonate, if in contact with it, such that precipitates on a free-flowing well are a result of a disturbed chemical equilibrium at the wellhead. The partial pressure of carbon dioxide there is 2.4 bar. When the tube of well BB-1 is fully “warmed up”, the artesian pressure is estimated at 2.9 bar, and knowing that there is 0.03 bar in the air, it is obvious that carbon dioxide is freed at the wellhead and causes supersaturation of calci- um carbonate in the solution and its precipitation (Papić 1991; Martinović & Milivojević 1998).Calcium carbon- ate precipitation within the zone of only 15 m from the well was attributed to a lowered total pressure and free- ing of carbon dioxide from the water, aided by elevated temperature. At Metković, well BMe-1, built in 1987, delivered mineral water at a temperature of 63 °C; there was no notable incrustation but biofilm was present at the point of discharge (Fig. 2). Incrustation and microbial pro- fig. 10: Classification of min- eral waters (a) and microorgan- isms (b) based on ph, including categorization of wells by type of mineral water and microbial community. Legend: BB-1– well in Bogatić; BMe-1 – well in Metković; BBe-1 – well in Belotić; RB-3/90 – well in Radaljska Banja; LB-4 – well in Lukovska Banja; LB-5 – well in Lukovska banja; Vg-2 – well in Vranjska Banja; Vg-3 – well in Vranjska Banja. fig. 11: Condition of well BB-1 (Photo: M. Lazić, 2014). VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 311 the formation of precipitates are: partial pressure of CO2, temperature, pH and TDS. The Langelier Stability Index was applied to determine the propensity of water to form a calcium carbonate precipitates. where: SI Lang – Langelier Stability Index, pH – measure of hydrogen ion (H+) activity in solution, pHs – pH at which the water is saturated with calcium carbonate, t – groundwater temperature (°C), Ca2+– calcium concentration (mg/l), HCO3 − – bicarbonate concentration (mg–ekv/l), S.O. – dry residue (mg/l). cesses (biofouling) accelerated well ageing and caused changes in the organoleptic properties and chemistry of the water. Microorganisms rely on the mineral substrate of the mineral water for their growth and survival. To remove the outcomes of undesirable processes, wells are general- ly rehabilitated by a combination of mechanical, chemi- cal and hydraulic treatments of the screen and wellhead region, with the goal of restoring the original well capac- ity (Fig. 14). During the two-year study, most of the wells exhib- ited precipitation of calcium carbonate (Fig. 12, Fig. 13). Consequently, the high content of CO2 and HCO3 − re- sulted in the formation of precipitates. The Langelier Saturation Index (LSI) and Ryznar Stability Index (RSI) were used to determine the incrustative and corrosive properties of water. The main parameters that lead to fig. 12: Encrustation sampling points: a) well BB-1, b) well LB-4, c) well LB-5 (Photo: M. Lazić, 2014-2016). fig. 13: Well encrustations: a) BB-1 at Bogatić, and b) LB-4 and LB-5 at Lukovska Banja (Photo: O. krunić, 2016). fig. 14: Rehabilitation of well BB-1 at Bogatić (Photo: M. Lazić, 2014). MINERAL WATERS OF SERBIA AND DEVELOPMENT OF PHOTOTROPHIC MICROBIAL COMMUNITIES NEAR POINTS OF ... ACTA CARSOLOGICA 46/2–3 – 2017312 If: SI Lang > 0, the water is prone to precipitation of CaCO3; SI Lang < 0, the water is not prone to precipi- tation of CaCO3, it is corrosive if oxygen is dissolved in water. The aggressiveness of water to metal surfaces was assessed on the basis of the Ryznar Stability Index (SI- Ryz): If: SIRyz > 8.5, the water is highly aggressive towards metal; 6.8. < SIRyz < 8.5 water is aggressive towards met- al; 6.2 < SI Ryz < 6.8 water is stable; 5.5 < SIRyz < 6.2 wa- ter is prone to lime precipitation; and SIRyz < 5.5 water is highly prone to lime precipitation. In the present case, the calculated Langelier Satura- tion Index (SILang) and Ryznar Stability Index (SIRyz) of the studied mineral water occurrences are shown in Tab. 4. The results presented in Tab. 4 corroborate that at a mineral water temperature above 60 °C, there was corro- sion-free incrustation – namely lime precipitation (SIL- ang > 0, SIRyz = 5.5). Consequently, the general conclu- sion was that corrosion of the wellhead and upper parts of the well casing was caused by the presence of free CO2 and H2S (Tab. 2). It is believed that microorganisms play the impor- tant role of biomineralizers in the CaCO3 precipitation process. The cyanobacterium produces CaCO3 by reduc- ing the bicarbonate ion (HCO3 −) to carbonate, whereby the medium is additionally alkalized and the carbonate ion (CO3 2−) created from the following reaction (Plum- mer et al. 1978; Buhmann & Dreybrodt 1987; Chafetz 1994; Flügel 2004): HCO3 − + OH−→ CO3 2− + H2O (4) Given a certain percentage of the Ca2+ ion in the studied occurrences of mineral water, CaCO3 is precipi- tated on the surface of cyanobacteria according to the reaction: Ca2+ + CO3 2−→CaCO3↓ (5) Although the role of microorganisms in the process of CaCO3 crystallization has not been completely under- stood, the biomineralization process is believed to take place at the cell wall and that extracellular macromol- ecules (extracellular polymeric substances – EPS) play a particularly important role in this process. Cyanobacte- ria can provide nucleation sites for the formation of min- erals and CaCO3 precipitates, on the sheaths, where they become trapped among the trichomes. As algae grow, high pH values favor the precipitation of calcium car- bonate that forms layers which remain stable for a long period of time (Ward et al. 2002). USE OF ALGAE IN BIOTECHNOLOGy Since the formation of minerals by geologic processes is a long-term process, the ability to stimulate microorgan- isms to create targeted biominerals is of great importance and it is possible to cultivate select algae and microor- ganisms from thermal ecosystems for that purpose. With the exception of calcium carbonate, many important biominerals can be obtained through biomineralization processes (i.e. carbonate oxides, sulfides, phosphates, sul- fates, nitrates, and silicate halogenides) (Plummer et al. 1978; Buhmann & Dreybrodt 1987; Chafetz 1994; Flügel 2004). A good example of the use of thermophilic algae in biotechnology is the production of bioethanol (Li et al. 2008) and the extraction of poly-hydroxybutyrate from Synechococcus spp., a compound used to degrade plastics (Mongra 2013), among other biorefinery products. Cosmarium laeve, which has been detected in Bogatić, Belotić, and Lukovska Banja, is of special in- terest since it is a natural source of various bioactive molecules. Phormidium sp., for example, can produce antimicrobial compounds (Mongra 2013). Extremely thermophilic species, such are those identified in Vran- jska Banja (VG-2 and VG-3), are used in biotechnology to produce vitamins, enzymes, lactic acid and other sub- stances important in agriculture and medicine. Also, Spirulina is a rich source of vitamins (A, C, D, E and B), minerals, fiber, beta-carotene, protein, and minerals (iron, potassium, magnesium, sodium, phos- phorus, zinc and selenium). It has a positive effect on the immune and nervous systems, brain and eyes, protects the body from harmful substances, helps reduce weight, and decreases the risk of cancer, all owing to a powerful antioxidant – zeaxanthin. It also has anti-inflammatory and anti-allergic properties and helps relieve stress and depression (Hoseini et al. 2013; Šaraba & Krunić 2017). tab.4: Langelier Saturation Index (SILang) and Ryznar Stability Index (SIRyz). Well pH pHs SILang SIRyz BB-1* 6.9 6.2 0.7 5.5 BMe-1 6.9 6.2 0.7 5.5 BBe-1 7.2 7.3 -0.1 7.5 RB-3/90 9.3 9.3 0 9.3 LB-4* 7.5 6.6 0.9 5.7 LB-5* 6.6 6.4 0.2 6.2 VG-2* 7.9 6.2 1.7 4.5 VG-3* 8.0 6.1 1.9 4.2 *BB-1, LB-4, LB-5, Vg-2, Vg-3 – occurrences of mineral water whose temperature was higher than 60 °C. VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć ACTA CARSOLOGICA 46/2–3 – 2017 313 The studied points of emergence of mineral water and wellheads in Serbia (Bogatić, Metković and Belotić in the Mačva District, as well as Radaljska Banja, Lukovska Banja and Vranjska Banja) were found to be favorable habitats for various groups of microorganisms. The re- sults served as a basis for drawing general conclusions about the biodiversity and geoenvironment, given that mineral waters are extracted from karst, fractured and intergranular aquifers of different lithologies and geolog- ic age. The water types were HCO3 − – Na++K+ and SO4 2−, HCO3 − – Na++K+, and the temperature range from 30 to 93.9 °C. The mineral waters extracted from igneous rocks exhibited the largest number of phototrophic microbial taxa: 15 species were found at Radaljska Banja and 12 in Vranjska Banja. There were ten phototrophic microbial taxa each in the karst aquifers within Triassic limestones and dolomites at Bogatić and Belotić, and nine taxa in the Neogene fractured sandstone aquifer at Metković. In Lukovska Banja, the mineral water is formed in sedimen- tary and metamorphic rocks (Cretaceous limestones, serpentinites, cherts), with occasional diabase interbeds, where the smallest number of phototrophic microbial taxa were noted (only eight). Given that sampling was conducted at the ground surface, apart from the chemi- cal composition of the water, microclimate played an im- portant role. The research revealed representatives of three groups of phototrophic microorganisms: Cyanobacteria, Chlorophyta and Bacillariophyta. Cyanobacteria were the most numerous and dominated by the order Oscil- latoriales, followed by representatives of the phylums Bacillariophyta and Chlorophyta. Phototrophic biofilms near points of emergence of mineral waters and at wellheads in Serbia had not been examined in the past. The present study, apart from ad- dressing the rehabilitation of biofouled wellheads by me- chanical, chemical and hydraulic treatments at Bogatić, is significant because it provides an assessment of the biodiversity of mineral waters in Serbia. The general conclusion was that mineral water oc- currences favor the development of unique microbial communities, which deserve special attention from the perspective of protecting the environment and genetic pool. Hence, protection of mineral waters is recom- mended to conserve biodiversity. CONCLUSIONS ACKNOWLEDGEMENT The Serbian Ministry of Science has financially supported this research under contracts TR33053 and OI176020. REFERENCES Aguilera, A., Souza-Egipsy, V. & R. Amils, 2012: Pho- tosynthesis in extreme environments.- In: Najaf- pour, M. (ed.) Artificial Photosynthesis. InTech, pp. 271−288, Rijeka. 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VLADIMIR ŠARABA, SLAĐANA POPOVIć, OLIVERA KRUNIć, GORDANA SUBAKOV SIMIć, ŽELJKO KLJAJIć & MILOJKO LAZIć