Abstract UDC 582.23:551.435.82:549.742.111(560) Elif Özlem Arslan- Aydoğdu, Yağmur Avci, Nahdhoit Ahama- da Rachid, Batu Çolak & Nihal Doğruöz-Güngör: Screening of bacteria in Yarık Sinkhole, Antalya, Turkey for carbonate dissolution, biomineralization and biotechnological poten- tials Abiotic and biotic factors, especially microorganisms, play a role in the development of cave formations and the existence of unique characteristics of each cave. Due to the ecological conditions that characterize the cave environments, highly specialized microorganisms that are the main source of diverse bioactive compounds, inhabit these environments. The aim of this study is to determine the role and biotechnological poten- tial of the bacteria isolated from Yarık Sinkhole located in An- talya (Turkey) by screening their ability to induce the CaCO3 precipitation, to hydrolyze urea, to induce calcite dissolution, and screening their possession of NRPS/PKS gene clusters. The most prevalent phylum is the Bacillota (synonym Firmicutes) (75.7 %), while the dominant species is Bacillus pumilus (33 %). All the isolates showed crystal formation on B4 agar me- dium, and the Energy dispersive X-Ray spectroscopy (EDS) analyses showed that the crystals are predominately composed of calcium, carbon and oxygen. Ninety-six (96 %) of our iso- Izvleček UDK 582.23:551.435.82:549.742.111(560) Elif Özlem Arslan- Aydoğdu, Yağmur Avci, Nahdhoit Aha- mada Rachid, Batu Çolak & Nihal Doğruöz-Güngör: Pregled bakterij v vrtači Yarık, Antalja, Turčija, v zvezi z raztapljan- jem karbonata, biomineralizacijo in biotehnološkim poten- ciali Abiotski in biotski dejavniki, zlasti mikroorganizmi, imajo pomembno vlogo pri nastanku jamskih oblik in glede edin- stvenih značilnosti vsake jame. Zaradi ekoloških razmer, ki so značilne za jamska okolja, ta okolja naseljujejo visoko spe- cializirani mikroorganizmi, ki so glavni vir raznih bioaktivnih spojin. Cilj te študije je opredeliti vlogo in biotehnološki po- tencial bakterij, izoliranih iz vrtače Yarık v Antalji (Turčija), s pregledom njihove zmožnosti, da povzročijo obarjanje CaCO3, hidrolizirajo sečnino, povzročijo raztapljanja kalcita, in nji- hove vsebnosti genskih skupin NRPS in PKS. Najpogostejše deblo je Bacillota (sinonim: Firmicutes) (75,7 %), prevladujoča vrsta pa je Bacillus pumilus (33 %). Pri vseh izolatih se je na agarnem gojišču  B4 pojavila tvorba kristalov, analize z en- ergijsko disperzivno rentgensko spektroskopijo (EDS) pa so pokazale, da so kristali sestavljeni pretežno iz kalcija, ogljika in kisika. Šestindevetdeset (96  %) naših izolatov ima nega- tivno ureolitično aktivnost. Glede na ta rezultat in zmožnost, ACTA CARSOLOGICA 52/1, 147-166, POSTOJNA 2023 DOI: https://doi.org/10.3986/AC.V52I1.10383 1 Elif Özlem ARSLAN- AYDOĞDU, Istanbul University, Faculty of Science, Department of Biology, 34134, Istanbul, Turkey, e-mail: eoarslan@istanbul.edu.tr 2 Yağmur AVCI, Istanbul University, Institute of Graduate Studies in Sciences, 34134 Vezneciler, Istanbul, Turkey, e-mail: ygmr.avci@hotmail.com 3 Nahdhoit AHAMADA RACHID, Istanbul University, Institute of Graduate Studies in Sciences, 34134 Vezneciler, Istanbul, Turkey, e-mail: nahamadarachid@gmail.com 4 Batu ÇOLAK, Istanbul University, Institute of Graduate Studies in Sciences, 34134 Vezneciler, Istanbul, Turkey, e-mail: batucolak4@gmail.com 5 Nihal DOĞRUÖZ-GÜNGÖR*, Istanbul University, Faculty of Science, Department of Biology, 34134, Istanbul, Turkey, e-mail: ndogruoz@istanbul.edu.tr * Corresponding author Prejeto/Received: 17. 8. 2021 SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS PREGLED BAKTERIJ V VRTAČI YARIK, ANTALJA, TURČIJA, V ZVEZI Z RAZTAPLJANJEM KARBONATA, BIOMINERALIZACIJO IN BIOTEHNOLOŠKIM POTENCIALI Elif Özlem ARSLAN- AYDOĞDU1, Yağmur AVCI2, Nahdhoit AHAMADA RACHID3, Batu ÇOLAK4 & Nihal DOĞRUÖZ-GÜNGÖR5* ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR 1. INTRODUCTION Caves are natural underground cavities accessible to hu- mans and are characterized by poor nutrients, low but constant temperature, and high humidity (Gillieson, 1996; Zada et al., 2021). The most common cave type on earth is the karstic cave (Bartolomé et al., 2021). The formation of caves starts with the dissolution of soluble rocks like gypsum and limestone under the chemical ac- tion of water. Under mechanical erosion, caves and cave structure are gradually forming (Becker et al., 2020; Ai et al., 2022). Formation and change of cave structure can be affected by the biogeochemical cycles in the cave environ- ment. These cycles are mediated by biotic factors such as microorganisms and abiotic factors like temperature and organic compounds. The composition of cave microbiota is controlled by different factors like the type of carbon sources and the specific minerals present inside the cave (Ai et al., 2022). Previous studies showed that microor- ganisms can participate in rock corrosion or in shaping cave structures. Mechanisms like biomineralization, dis- solution of minerals and degradation of organic com- pounds, in which microorganisms use enzymes and their extracellular biological compounds, have been reported (Cañaveras et al., 2001; Jones, 2001; Ríos et al., 2011). The oligotrophic characteristic of the cave environment is not an obstacle to colonization and dominance of microbes that constitute the primary producers in these dark, un- derground ecosystems. It is reported that chemoautotro- phic microbes, common in caves, fix carbon, maintain the energy cycle in caves, and sustain the life of many other cave organisms (Wu et al., 2015). Moreover, some chemoautotrophic caves like Movile and Frasassi Caves, investigated for their microbial diversities with biogeo- chemical reactions, were observed with microorganisms including sulfur oxidizers, CO2 fixers, methanotrophs, and nitrifying microorganisms (Jones et al., 2008; Jurado et al., 2020; Chiciudean et al., 2022). Researchers are mostly interested in studying the cave microbiota due to their ability to adapt to under- ground living conditions, which have poor nutrients and a lack of light. The deposition of nitrate/carbonate, the breakdown/dissolution of speleothems and cave walls, as well as other chemical reactions are observed in these areas (Johnston et al., 2021). The precipitation or dis- solution of nitrate and carbonates has been reported to be passive; microorganisms produce enzymes or behave like nucleation sites that mediate the microbial-induced precipitation or dissolution processes (Zada et al., 2021). Calcium carbonate precipitation is a good example of microbially extracellular biomineralization. Microbially induced CaCO3 precipitation (MICP) results from the metabolic interactions between diverse microorganisms and organic and/or inorganic compounds present in the environment (Sarda et al., 2009; Qian et al., 2010). This process is known to occur through various metabolic pathways such as photosynthesis, sulfate reduction, an- aerobic sulfur oxidation, and urea hydrolysis (Sarda  et al., 2009; Qian et al., 2010). Microorganisms that induce the precipitation of calcium minerals have been isolated from different ecosystems including soil (Boquet  et al., 1973), sea sediments (Turchyn et al., 2021), active an- aerobic mud (Su & Yang, 2020), and caves (Baskar, 2009; Miller et al., 2018).  MICP is used with great interest in different areas like the remediation and restoration of various building materials (Ortega-Villamagua et al., 2020), construction of bio-concrete (Su et al., 2021), removal of contaminants from groundwater and soil (bioremediation) (Dhami et al.,  2013), heavy metal removal from wastewater (Tya- gi  et al., 2020), bio self-healing dental composite (Sei- fan et al., 2020), and technology of microbially enhanced petroleum recovery (MEROR) (Saravanan et al., 2020). The most studied bacteria in the MICP are shown in the literature to have the ability to hydrolyze urea. However, bacteria with urease negative activity with the ability to lates have negative ureolytic activity. According to this result and having the ability to induce the CaCO3 precipitation, bac- teria in this environment use other biosynthesis pathways than urea hydrolysis. MgCO3 and CaCO3 were dissolved by 61 % and 59 % of the isolates, respectively. In addition, 5.9 % and 53.7 % of the isolates showed the possession of PKS and NRPS genes, respectively. This result reveals that our isolates have high in- dustrial and biotechnological potential. They may constitute good candidates for further biotechnological applications such as construction of bio-concretes, bioremediation, soil fertility, and production of biologically active secondary metabolites. Keywords: cave bacteria, non-ureolytic bacteria, carbonate dissolution, carbonate precipitation, polyketide synthase, non- ribosomal peptide synthetase. da povzročijo obarjanje CaCO3, bakterije v tem okolju upora- bljajo druge načine biosinteze, ne hidrolizo sečnine. MgCO3 in CaCO3 je raztopilo 61 % oziroma 59 % izolatov. Poleg tega je 5,9 % in 53,7 % izolatov imelo gene genskih skupin PKS ozi- roma NRPS. Ta rezultat kaže, da imajo naši izolati velik indus- trijski in biotehnološki potencial. Lahko so zelo primerni za nadaljnjo biotehnološko uporabo, na primer za pripravo bio- betona, bioremediacijo, rodovitnost tal in proizvodnja biološko aktivnih sekundarnih metabolitov. Ključne besede: jamske bakterije, neureolitične bakterije, razta- pljanje karbonata, obarjanje karbonata, poliketidna sintaza, neri- bosomalna peptidna sintetaza. ACTA CARSOLOGICA 52/1 – 2023148 SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS induce the calcium precipitation process should not be ignored (Türkgenci & Doğruöz Güngör, 2021). An opposite process of calcium carbonate precipi- tation in the cave is the weathering of carbonate rock, cave walls, and sediments. The dissolution pits are regu- larly observed on rock surfaces in caves. These pits are formed by the dissolution and decomposition of rocks. The breakdown of rocks is the result of biological action and inorganic reactions such as condensation, corro- sion, and erosion. Microorganisms can produce organic acids which alter the pH of the environment and result in the dissolution of the calcium carbonate (Cañaveras et al., 2001; Jones, 2001; Johnston et al., 2021). On the other hand, microorganisms can produce enzymes that cata- lyze the reaction of CO2 and water releasing ions that in- duce calcite dissolution (Li et al., 2006; Johnston et al., 2021). The cave microorganisms that cause the dissolu- tion of MgCO3 and CaCO3 may have wide use in soil re- mediation and soil fertility in areas affected by high salt concentrations (Orhan et al., 2017). Previously, it showed that cave microorganisms can act either as carbonate weathering or/and precipitation inducers. In this fact, it is important to study these two features in the same mi- croorganisms to select the most suitable microorganisms to be used for industrial and biotechnological purposes. In addition to the microbially-mediated carbonate dissolution and precipitation processes, other biologi- cal processes are occurred in cave environments. These processes result to the production of biological com- pounds that could be used for pharmaceutical, food, and other biotechnological and industrial applications. Polyketides (PKs) and non-ribosomal peptides (NRPs) are two bioactive compound groups that have important roles because of their various biological activities. Due to the oligotrophic nature of the caves, PKs and NRPs can give their producers an advantage over competitive mi- croorganisms (Bauer et al., 2018; Bukelskis et al., 2019; Lukoseviciute et al., 2021). PKs synthesized by bacteria are mostly encoded by the Polyketide synthase (PKS) gene cluster or the hybridization of PKS and nonribo- somal peptide synthetase (NRPS) gene clusters. These compounds are known for their wide uses in agriculture (plant growth/antimicrobial substances) and medicine (Olishevska  et al.,  2019). Lipopeptide bio-surfactants synthesized by non-ribosomal peptide synthetases (NRPSs) have various roles such as antimicrobial, anti- tumoral, antioxidants, iron acquisition, immunosuppres- sive, insecticidal, nematocidal, and phytotoxic activities (Sukhanova  et al.,  2018; Bukelskis  et al.,  2019; Lukos- eviciute et al., 2021).  The geochemical characteristics of cave environ- ments vary from one cave to another and make the unique characteristic of each cave. Since the microbial composition of an environment is also dependent on the local physicochemical parameters, the microbial diver- sity of caves follows the same rule of cave uniqueness. Therefore, our study aimed to investigate the importance of bacteria isolated from Yarık Sinkhole in the formation of caves and cave structures, as well as their potential uses in industrial/biotechnological areas. In this fact, the MICP and CaCO3/MgCO3 weathering abilities of isolates were studied for determining some of their roles in cave formation and changes of cave structure. In addition, the urease activity was screened to determine if the isolates from this sinkhole exhibiting calcium carbonate precipi- tation are using the urea hydrolysis pathway. Moreover, to reveal the potential of the Yarık Sinkhole’s bacteria to synthesize bioactive compounds, PKS and NRPS gene clusters were screened in these isolates.  In the last, the isolates suitable for use within the framework of our pur- poses for future industrial and/or biotechnological appli- cations were stocked. 2. MATERIALS AND METHODS 2.1. CAVE BACKGROUND Our studying area was the Yarık Sinkhole (GPS coordi- nate: UTM 448068.47 E 4036006.77 N) which is locat- ed on the Sivastı Plateau centred 30 km in the north of Gazipaşa in Antalya-Turkey. Yarık Sinkhole has a total length of 1378 m and 533 m in depth. At the first en- trance, the cave was explored up to a depth of -300 m and reached a depth of -533 m in 2016. The watershed of the cave is a closed valley where the main rock is limestone with little sediment in the basin of the valley. The entrance of the cave is a broad opening and has a vertical type, occasional narrow passages and a stepped descent down and a shallow pond formation at the base. Generally, the Yarık Sinkhole’s ceiling is higher, but some of the narrow passages are difficult to pass, especially in a flood situation when these passages will be blocked. Unlike most vertical caves, in the Yarık Sinkhole speleo- thems such as flowstones and cave pearls are found in the horizontal portion. ACTA CARSOLOGICA 52/1 – 2023 149 2.2. SAMPLING AND ISOLATION OF MICROORGANISMS Soil and surface samples were taken from the depth -80 m and -320 m during the cave exploration in 2014. About 25 g of each soil samples were taken under aseptic con- ditions by using sterile disposable sample container with screw cap. The surface samples were taken with sterile swab from the cave walls (10 cm2 wide area) located near the soil sampling areas. All samples were transported un- der refrigerated conditions to the laboratory within 24 hours. After reached to laboratory, samples were instantly diluted in a serial dilution range of 10-1 -10-5 and 100 µl from each dilution was inoculated on Reasoner's 2 Agar (R2A) and TSA (Tryptic Soy Agar – prepared in 1/2 ratio) plates in triplicate and incubated at 28°C for 7 days. Plates were daily checked and colonies with different morphol- ogy were selected, Gram-stained, and purified. Purified colonies were stored at -86 oC for subsequent uses. 2.3. GENOMIC DNA ISOLATION, PCR AMPLIFICATION, AND SEQUENCING The genomic DNA of the isolates was isolated by using the bacterial ExgeneTM Cell SV DNA isolation Kit (Ge- neAll®). The 16S rRNA gene of each isolated DNA was amplified through polymerase chain reaction (PCR) by using the universal primers 27F (5'-AGAGTTT- GATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTT- GTTACGACTT-3'). The PCR mixture was prepared with a volume of 50 μL containing 20 nM of each primer, 10 ng of DNA, 2.5 U of Taq DNA polymerase (Takara Bio) and run in a single block thermal cycler (BIORAD). The reaction was performed under the following conditions: 1 min at 95°C for initial DNA denaturation, followed by 35 cycles composed of second DNA denaturation for 15 s at 95°C, annealing step at 55°C for 15 s, and 10 s of exten- sion step at 72°C, then a final extension step performed at 72°C for 10 min. The PCR products were sequenced by the Sanger sorting method and sequences were read by the ABI 3130 Sequencer (Applied Biosystems). The 16S rDNA sequences were deposited in GenBank (NCBI) under the accession numbers MZ646235- MZ646264, MZ646266- MZ646299, MZ420151, MZ821657. 2.4. DETERMINATION OF UREASE ACTIVITY AND CACO3 PRECIPITATION OF CAVE BACTERIA All the isolates were inoculated in Christensen's broth (1 liter of the medium contains: Peptone (1.0 g); Glucose (1.0 g); Di-sodium hydrogen-phosphate (1.2 g); Potas- sium dihydrogen phosphate (0.8 g); Sodium chloride (5.0 g); Phenol red (0.004 g) and 10 ml of urea solution) for urease activity analysis. A change in color after 2-3 days of incubation at 28°C was recorded as urease-positive re- action (Omoregie et al., 2018). The screening of the bacterial isolates for crystal- forming bacteria was performed with calcification pro- moting agar medium B4 composed of calcium acetate (0.25 g), yeast extract (0.4 g), agar (1.5 g), and, glucose (1.0 g) in 100 ml distilled water with pH 8.0. The isolates ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR Figure 1: The geographical location of Yarık Sinkhole in Antalya, Turkey (http://www.google.com.tr/maps/place., Reviewed on: May 30, 2021). ACTA CARSOLOGICA 52/1 – 2023150 were inoculated on the B4 agar medium, incubated at 28oC, and controlled under a light microscope every day for a period of 28 days, then crystal-forming ones were recorded (Boquet et al., 1973). Non-inoculated plates were used as negative controls. 2.5. CRYSTAL ANALYSIS The crystal morphologies of three isolates selected based on their species were observed using Scanning Electron Microscopy (SEM). Moreover, analysis of elemental composition was performed using energy-dispersive X- ray spectroscopy (EDS). First, the crystals were purified from the B4 agar medium by cutting out agar pieces and transferred to a 50 ml Falcon tube. Then the agar pieces were dissolved using a microwave oven. Both the super- natants and sediments were twice washed with 15 ml sterile distilled water until the agar was removed. As de- scribed by Marvasi et al. (2012) with some modifications, the crystals were collected and distributed into sterile empty plates and stored at 28°C for 7 days for drying. SEM-EDS analyses of crystal formations were performed in the physics laboratory of Istanbul University by using an EDAX Octane detector equipped with FEI Versa 3D Dual Beam and Dual Beam. The visual appearance mag- nifications were ranging from 330 to 12000 times. 2.6. DETERMINATION OF CACO3 AND MGCO3 WEATHERING POTENTIAL OF ISOLATES Deveze-Bruni’s CaCO3 agar (per liter: glucose (20 g); NaCl (10 g); MgCl2 (3 g); MgSO4.7H2O (0.5 g); KCl (0.4 g); (NH4)2SO4 (0.2 g); agar (15 g) and CaCO3 (10 g) and Deveze-Bruni’s MgCO3 agar (per liter: glucose (20 g); NaCl (10 g); MgCl2 (3 g); MgSO4.7H2O (0.5 g); KCl (0.4 g); (NH4)2SO4 (0.2 g); agar (15 g) and MgCO3 (10 g) were used for the investigation of CaCO3 and MgCO3 dissolu- tion potential of isolates (Cacchio et al., 2004; Orhan et al., 2017). The bacterial isolates were incubated at 30oC for 2–3 weeks on Deveze-Bruni’s CaCO3 agar and De- veze-Bruni’s MgCO3 agar media. The formation of a clear zone around the bacterial growth indicates the dissolu- tion of CaCO3 or MgCO3 minerals. It should noted that the media used in this experimentation are opaque and become transparant after the dissolution of the minerals ( CaCO3 or MgCO3 ) containing the media. 2.7. SCREENING OF PKS AND NRPS GENE CLUSTERS IN BACTERIAL ISOLATES For the PCR amplification of PKS and NRPS genes, three sets of primers were used: DegKS2F/DegKS2R for PKS, A3F/A7R and MTF/MTR for NRPS (Table 1). The components and reaction conditions of the PCR mixture are as follows: PKS DegKS2F/DegKS2R (25 μL): 1 μL template, 12.5 μL EmeraldAmp® MAX HS PCR Master Mix (Takara Bio), 5% of DMSO and 2.5 μL each primer (5μM); the PCR conditions were composed of initial denaturation of 5 min at 94°C, followed by 40 cycles of 40 s at 94°C, 40s at 50°C, and 75s at 72°C, and the final extension was at 72°C for 5 min. NRPS MT-F/MT-R (25 μL): 1 μL template, 12.5μL Em- eraldAmp® MAX HS PCR Master Mix (Takara Bio), 2.5 μL each primer (5μM); initial denaturation was at 94ºC for 3 min, followed by 35 cycles of 60 s at 94ºC, 60 s at 55ºC and 2 min at 72ºC and the final extension step was at 72ºC for 7 min. A3F/A7R (25 μL): 1 μL template, 12.5 μL Emeral- dAmp® MAX HS PCR Master Mix (Takara Bio), 5% of DMSO, and 2.5 μL each primer (5μM); initial denatur- ation was 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 59°C and 90 s at 72°C, followed by a 5-min extension at 72°C. After the polymerase chain reaction for the PKS and NRPS targeting genes, PCR products were run in a 1% agarose gel through electrophoresis. SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS Table 1: Primers and PCR cycling conditions were used in this study. Primer Sequence Reference Polyketide synthase DegKS2F 5’-GCIATGGAYCCICARCARMGIVT-3’ (Amos et al., 2015) DegKS2R 5’-GTICCIGTICCRTGISCYTCIAC-3’ (Amos et al., 2015) Non-ribosomal peptide synthetase MT-F 5’-GCNGGYGGYGCNTAYGTNCC-3’ (Tambadou et al., 2014) MT-R 5’-CCNCGDATYTTNACYTG-3’ (Tambadou et al., 2014) A3F 5’-GCSTACSYSATSTACACSTCSGG (Amos et al., 2015) A7R 5’- SASGTCVCCSGTSCGGTA (Amos et al., 2015) ACTA CARSOLOGICA 52/1 – 2023 151 3. RESULTS At the end of the incubation, 70 colonies (33 from soil and 37 from surface samples) growing in R2A and ½ TSA media with different morphological characteristics were isolated. The identified isolates are shown in Figure 2 and Table 2 (supplement). Each amplified fragment of the 16 rRNA gene of each isolate was sequencing, blast- ed in the NCBI platform. Species in the NCBI platform showing high identity to our isolates were chosen for naming our isolates. When evaluated based on sample diversity, 33% of our isolates were isolated from the soil sample 1S, 24% from the surface sample 1Y, 20% from the soil sample 2S and 23% from the surface sample 2Y. The obtained isolates belong to 11 different genera and 16 different species 75.7 % of the isolates belong to Bacil- lota (synonym Firmicutes), while 12.9 % and 5.7 % be- long to Pseudomonadota (synonym Proteobacteria) and Actinomycetota (synonym Actinobacteria), respectively. The distribution of our isolates was observed with domi- nance of the strains belonging to Bacillus genus (73%) of which the most common species (33%) was observed as B. pumilus of which 57% of them were isolated from soil samples (Table 2 (supplement) and Figure 2). The sec- ond dominant species is B. safensis (19%) of which 62 % of them were observed from the soil samples. It was fol- lowed by Bacillus sp. (13%) of which 78% were isolated from the soil samples (Table 2 (supplement) and Figure 2). The less common strain of Bacillus genus observed in our study is B. zhangzhouensis (Figure 2). 8% of iso- lates belonging to Bacillus genus were isolated only from sampling area 2 (-320m) consisting of 1 soil sample and 3 surface samples. After Bacillus, the second most common genus in this study was the Brevundimonas with only the spe- cies Brevundimonas vesicularis was observed (Table 2 in supplement). Most of our isolate belonging to this spe- cies are isolated from surface sample Y2. The results pre- sented in Table 2 (supplement) show that these isolates differ from each other in terms of their phenotypic and genotypic characteristics. 3.1. UREASE ACTIVITY AND CACO3 PRECIPITATION OF CAVE BACTERIA All isolates except Y112 and Y129 (Table 2 supplement) showed color change of the urea medium from orange to pink color. This observation suggests the production of urease enzyme, which hydrolyzes the urea present in the medium. Under the light microscopy, crystals were observed on all the isolates inoculated on the B4 agar medium (Table 2 supplement). The crystal formation is the result of the precipitation of minerals on the surface of the bacteria. It was observed that 23 %, 6 %, 17 % and 54 % of these isolates formed crystals within 1-3 days, 4-7 days, 8-14 and 15-24 days respectively (Table 2 supple- ment). Moreover, 75% of the isolates that form crystals in 1-3 days belong to Bacillus (Table 2 supplement). The other identified genera observed with crystal formation within 1-3 days are Sphingomonas, Staphylococcus and Stenotrophomonas (Table 2 supplement). As seen in Table 2 (supplement), the characteristics of the isolates differ although they are the same species. For example, the iso- lates Y108, Y113, Y124, and other isolates that are identi- fied as B. pumilus (Table 2 supplement) shown different features in terms of calcium carbonate precipitation. 3.2. CRYSTAL MORPHOLOGIES AND ELEMENTAL COMPOSITION Our isolates were observed daily under light microscopy for their potential to induce the formation of crystals on B4 agar medium. Most of the crystals formed are square and rectangular. However, other forms of crystals like spherical, ellipse, hexagonal, and broken rice are also observed in our study (Figure 3). Under the light micro- scope, crystals formed by most of our isolates (83 %) are away from their colonies (Table 2 in Supplement). No crystal formation was observed on the negative control plates. Crystals observed in some isolates like Y178 (Fig- ure 3 (A)) have regular shape while those that have been observed from isolates like Y 208, Y271 (Figure 3 (B and C)) are of different shapes (spherical, broken rice, rectan- gular and square shapes). The crystal formed by three selected isolates (dif- ferent genera) were purified from the B4 agar medium and were analyzed under electron microscopy for their morphology observation. Spherical and ellipsoidal crys- tal shapes are noted in (Figures 4, 5, 6). In addition, EDS ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR Figure 2: Percentages of culturable heterotrophic aerobic bacteria isolated from the Yarık Sinkhole. The ‘others’ represent the per- centage of non-determined and species represented by less than 2 isolates. ACTA CARSOLOGICA 52/1 – 2023152 SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS Figure 3: Shapes of crystals produced by different isolates on B4 agar medium under light microscopy (E100- NIKON, ×40 for A, B, D and E; ×100 for C). A) Rectangular form of Crystals produced by Sphingomonas mucosissima (Y178) in B4 agar medium. B) Square and spherical forms of crystals produced by Staphylococcus epidermidis (Y208) on B4 agar medium. C) Different shapes of crystals (ice broke, rectangu- lar, irregular) observed on the growth area of Bacillus safensis (Y271) as well as on the other parts of the B4 agar plate. D) Square shape of crystals formed by Brevundimonas vesicularis (Y172) on the B4 agar medium. E) Square and rectangular forms of crystals produced by Bacillus pumilus (Y131) inoculated on B4 agar medium. F) Square and rectangular forms of crystals produced by Micrococcus aloeverae (Y147) inoculated on B4 agar medium. Figure 4. EDS spectrum and SEM analysis images of the calcium carbonate crystals produced by Stenotrophomonas maltophilia (Y116). A, B, and C) SEM observation of purified sphere-like shapes crystals formed by the Y116 isolate on the B4 agar medium. D and E) EDS spectrum results show the elemental composition of the calcium carbonate crystals produced by Stenotrophomonas maltophilia (Y116). ACTA CARSOLOGICA 52/1 – 2023 153 ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR Figure 5: EDS spectrum and SEM images of the calcium crystals produced by Micrococcus luteus (Y154). A and B) SEM observation of purified ellipsoid and spherical-shaped crystals formed by the Y154 isolate on the B4 agar medium. C) SEM observation of purified ellip- soid-shaped crystals formed by the same isolate. D and E) EDS spectrum results show the elemental composition of the calcium carbonate crystals produced by Micrococcus luteus (Y154). Figure 6 : EDS spectrum and SEM images of the calcium crystals produced by Sphingomonas mucosissima (Y178). A and B) SEM observa- tion of purified ellipsoid and spherical-shaped crystals formed by the Y178 isolate on the B4 agar medium. C) SEM observation of purified ellipsoid-shaped crystals formed by the same isolate. D and E) EDS spectrum results show the elemental composition of the calcium car- bonate crystals produced by Sphingomonas mucosissima (Y178). ACTA CARSOLOGICA 52/1 – 2023154 analyses confirmed that the crystals were composed pre- dominantly of calcium, carbon, and oxygen, suggesting precipitation of calcium carbonate and not of any other compound (Figure 4, 5, 6). The EDS on the isolate Y116 revealed that the atomic percentages of O, C and Ca were 55.66, 29.85, and 14.4%, respectively (Figure 4). When the crystals formed by the Y116 isolate are examined by SEM, it is observed that they have sphere-like shapes with an average length of 30 µm and a diameter of 2412.8 µm2 (Figure 4). The EDS on the isolate Y154 revealed that the atom- ic percentages of O, C, Ca, Na, Mo, and P were respec- tively 43.85 %, 47.05 %, 7.91 %, 0.75 %, 0.06 %, and 0.37 % (Figure 5). These results showed that the precipitate is composed of low concentrations of Na, K, Mo and Ca and high concentrations of O and C. Considering the SEM images (Figure 5), the ellipsoid and spherical- shaped crystals formed by Y154 have an average length of 19.10 µm and 21.7 µm, respectively. In addition, the SEM analysis of these crystals showed that the ellipsoid crystals have a surface area of 1074.91 µm2 while the spherical-shaped crystals have a surface area of 1478.2 µm2(Figure 5). According to the EDS results shown in Figure 6, the precipitate formed by Sphingomonas mucosissima (Y178) is formed mainly of O, C and Ca with their respective atomic percentages of 55.83 %, 21.43 % and 21.32 %, and a small amount of Mo, K and chlorine. It is seen that the crystals formed by this isolate are also ellipsoid and spherical. According to the SEM images, crystals with el- lipsoidal forms have an average length of 37.3 µm and a surface area of 3426.86 µm2, while those with spherical shapes have a radius of 20.65 µm and a surface area of 5358.58 µm2. 3.3. MGCO3 AND CACO3 DISSOLUTION POTENTIALS OF THE ISOLATES It was determined that 61 % and 59 % of the total iso- lates shown clear zones on the Deveze-Bruni’s CaCO3 agar and Deveze-Bruni’s MgCO3 agar media respectively. These observations show the ability of these isolates to dissolve MgCO3 and CaCO3, respectively. The ratios of bacteria that have MgCO3 and CaCO3 dissolution poten- tial are given in Table 3, according to their isolated areas. 37 % of the 70 strains were observed with a disso- lution of both MgCO3 and CaCO3, by showing a trans- parent zone around their growth, while 17 % did not show clear zones on the two media (Table 2 supplement). Despite the low abundance rate of Brevundimonas ve- sicularis in our study, all of our isolates belonging to this species showed dissolution ability of MgCO3 and 75 % of them dissolved the CaCO3. In this study, 34 and 29 identified Bacillus species SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS Figure 7: A) CaCO3 dissolution potential of isolates: Bacillus safen- sis (Y271), (Bacillus sp. Y177); B) MgCO3 dissolution potential of isolates: Bacillus pumilus (Y108), Bacillus pumilus (Y196). The clear zones observed around the bacterial growth suggest the disso- lution of calcium and magnesium carbonates present in the media. Table 3: Percentage of isolates able to dissolve carbonate com- pounds. Depth Sample type MgCO3 (%) CaCO3 (%) Both (%) 80 m Soil 83 91 78 Surface 76 65 53 320 m Soil 29 64 7 Surface 44 69 38 were able to dissolve MgCO3 and CaCO3, respectively. In our study, the most dominant isolate B. pumilus had MgCO3 and CaCO3 dissolution rates of 45 % and 38 %, respectively, while the MgCO3 and CaCO3 dissolution rates of B. safensis were respectively 19 % and 15 % (Table 2 supplement). In addition, the ability of the isolates to dissolve MgCO3 varies according to their isolation depth. Bacteria isolated fron -320 metres showed low MgCO3 dissolution rate than those that are isolated from -80 me- tres (Table 3). 3.4. SCREENING OF PKS AND NRPS GENES IN BACTERIAL ISOLATES In our results, we observed that 64 % of the bacteria screened for their handling of PKS and NRPS gene re- gions, contain at least one of these gene regions (Table 2 in supplement). We should note that only Y168, Y169, Y184 and Y208 isolates have been observed after elec- trophoresis with a fragment of 350 base pairs (bp) of the amplified PKS gene fragment. In addition, we observed ACTA CARSOLOGICA 52/1 – 2023 155 amplification of the NRPS gene region in 53.7 % of our isolates. However, most of them are amplified through while the A3R/A7F primer pair amplified fragments of 480 bp observed after electrophoresis. It is interesting to note that the isolate Y184 contains both PKS and NRPS gene amplified fragments (Table 2 supplement). The characteristics of the isolates according to the screened genes are shown in Table 2 (supplement). 4. DISCUSSION Reducing chemosynthetic materials, such a synthetic ce- ment, hydrocarbons and synthetic fertilizers in the envi- ronment may be achieved by replacing them with natural compounds. However, due to the hard laboratory condi- tions during the discovery and production of such natu- ral compounds from plants or animals, microorganisms become the favorable biological source of most bioac- tive compounds. In this context, new investigation areas screen for a high probability of new product discovery. One of the least investigated ecosystems in the world is the cave ecosystem. Due to the uncommon conditions that characterize these ecosystems, cave microorgan- isms, which are considered as successful bioactive com- pounds producers, are frequently studied to determine their biotechnological and industrial potentials (Hamedi et al., 2019; Teodosiu et al., 2019; Çandıroğlu & Doğruöz Güngör, 2020). In our study, the most identified isolates belong to Bacillota (synonym Firmicutes) (75.7%), especially the Bacillus genus. Similar to these results, the most domi- nant bacteria isolated from the Kadıini Cave belonged to Bacillota (synonym Firmicutes) (85%) and Bacillus pum- ilus was also the major Bacillus species identified in this cave (Doğruöz Güngör et al., 2020). On other hand, Bac- illota (synonym Firmicutes) (32.7%), was observed as the second dominant bacterial phylum in the cultured bacte- ria isolated from Dupnisa Cave (Türkgenci & Doğruöz Güngör, 2021). This bacterial phylum, known for its re- sistance to stress conditions like desiccation and environ- mental poor nutrient is frequently identified in extreme environments (Slepecky & Hemphill, 1992). Since each cave is unique in terms of morphological and geochemi- cal features, differences are also shown in its microbial communities. Generally, Bacillus, Streptomcyes, Clostrid- ium, Kocuria, Pseudomonas, Microbacterium, Paenibacil- lus, Brevibacillus, Sphingomonasi, and Stahylococcus are the most identified bacteria in cave environments (Laiz et al., 1999; Canaveras et al., 2001; Ikner et al., 2007; Her- zog Velikonja et al., 2014). In our study, we observed that all selected cave iso- lates have precipitated calcite on the B4 agar medium on different incubation days. In addition, the EDS spectra (Figures 4, 5, 6) showed Ca, C, and O peaks that could be qualitatively correlated with CaCO3. These results are similar to those observed in the study of Seifan et al. (2016) which determined the EDS spectra of pure cal- cium carbonate. This result confirms like other studies (Boquet et al., 1973; Cacchio et al., 2003) that many bac- teria can form CaCO3 crystals, especially in carbonate- rich environments such as karstic caves. We observe that calcium carbonate precipitation is an important physi- ological mechanism against bacterial calcium toxicity, which constitutes a vital key, especially for bacteria colo- nizing high calcium concentrations caves (Banks et al., 2010; Cacchio & Del Gallo, 2019). The key roles of mi- croorganisms in these processes have recently begun to be recognized. As previously noted, the microbially induced calci- um carbonate precipitation (MICP) is achieved through a different pathway. In general, it occurs with the in- crease in pH of the environment at the end of common metabolic processes of microorganisms such as carbon fixation, ureolysis, denitrification, ammonification, sulfate reduction, and methane oxidation (Fujita et al., 2000; Dupraz et al., 2004; Reeburgh, 2007; Van Paas- sen et al., 2010). It is also known that microorganisms, apart from these metabolic processes, produce extracel- lular polymeric substances that bind and condense cal- cium or other ions (Tourney & Ngwenya 2009; Ercole et al., 2012). Indeed, Ercole et al. (2007) showed that EPS isolated from B. firmus and B. sphaericus caused calcite precipitation. However, the medium (B4) used for cal- cium precipitation analysis in our study does not share properties for ureolysis, denitrification, ammonification, sulfate reduction, and methane oxidation. Therefore, it was not possible with our used method to conclude that the isolates are producing calcite precipitation through the EPS production. The results of the urease analysis test performed in our study also showed that the main way taken by most of our isolates for the calcite precipitation is far to be the urea hydrolysis. Lee et al. (2017) suggested that their isolates have used the deamination of the yeast extract present in the medium as a way of the MICP. Even though the B4 medium contains yeast extract which can be the substrate of the deamination, further experimen- tations will be required to determine the exact metabolic ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 52/1 – 2023156 pathway used by our isolates in the precipitation of the CaCO3. However, the yeast extract in the B4 medium has an important effect as a source of amino acids. In this way, bacteria that can show MICP activity through ureo- lytic pathway using urea released by arginine decarboxyl- ation can also be monitored in this medium. Although bacteria using the urea hydrolysis path- way are predominantly selected in microbial calcium car- bonate precipitation studies, especially those involved in biotechnology fields, we observed that 96% of our cave isolates precipitate the calcium carbonate without us- ing the ureolytic pathway. In this fact, the use of urease- negative bacteria in these areas should not be ignored. In addition, ureolytic bacteria can cause both environmen- tal and social health problems. Due to the production of ammonia/ammonium and nitrate at the end of the urea hydrolysis, using bacteria which use this way for calcium carbonate production in self-concrete repairing can im- pact human and animal health (Lee et al., 2017). The production of self-healing concrete materials is one of the important activities where MICP potential bacteria are used. The most used bacteria in this field are those with positive urease activities. However, these bac- teria can also induce the deterioration of the concrete. The ammonium produced as a by-product of urea hydro- lysis can act as a mild acid in alkaline environments. It re- acts with the hydroxide ions and forms ammonia inside the concrete; the ammonia will be oxidized and produce nitric acid which in turn will react with the precipitated CaCO3 to form a highly soluble component called calci- um nitrite. The dissolution of this component may cause the deterioration of the concrete matrix (Ganendra et al., 2014; Farhadi et al., 2020). Strains of Bacillus and Pseudomonas were observed through biochemical and biomolecular analyses as the most calcifying strains (Shirakawa et al., 2011; Banerjee & Joshi, 2016). In our previous study, strains belonging to the genus Bacillus isolated from Dupnisa Cave were the most calcifying isolates (about 30%) identified in this cave. Rivadeneyra et al. (1994) have previously shown that Bacillus sp. plays a major role in the carbonate depo- sition in natural habitats. Bacteria suggested to be used in the production of bio-concrete (self-healing concrete) should be resistant to harsh conditions such as high pres- sure during the bio-concrete production. In this context, spore-forming bacteria are the best candidates and most of them belong to the genus Bacillus (Reddy et al., 2020). On the other hand, although spore-forming bacteria have shown more advantages, non-spore-forming bac- teria have also been shown to crack repairing activities in some previous studies (Bansal et al., 2016; Choi et al., 2021). In our study, both spore-forming and non-spore- forming bacteria have been identified and we suggest that strains like Y108, Y109, Y110, Y122, Y146, Y181, Y193, Y212, and Y213 (Table 2 supplement) which are induc- ing the CaCO3 precipitation, spore-forming at the same time non-ureolytic bacteria may be good candidates in the production of bio-concrete. In addition, the strains Y116, Y178 which are non-spore-forming bacteria (Table 2 supplement) may have potential uses in the repair of concrete cracks by injecting or spraying them into the opening cracks. MICP is also considered one of the most effective and efficient methods for the removal of heavy metal pol- lutants from soil and groundwaters polluted by industrial wastes. Rajasekar et al. (2021) have determined that B. pumilus, which precipitates CaCO3, removed high (60- 75%) Cr, Zn, and Cu rates from polluted soil. Most of the bacteria used in our study belong to the genus Bacillus and precipitates CaCO3. We suggest that they may be eas- ily benefited in these areas. On the other hand, Hammes et al. (2003) used ureolytic bacteria to remove excess cal- cium from industrial wastes and found that 85-90% of soluble calcium has been precipitated as calcium carbon- ate in the treatment reactor. Considering this result, they suggested that problems such as clogging of pipes, heat exchangers, and failure of systems caused by the presence of high Ca2+ in industrial systems would be prevented (Hammes et al., 2003). Based on our results, we suggest that the CaCO3 precipitating isolates, both ureolytic and non-ureolytic bacteria, may also be used to solve such important problem of pollution It was determined that the crystal formation peri- od on the B4 medium, of the isolates used in our study differed at the strain level even if the isolates are of the same species. It may be important to pay attention to this parameter in biotechnological or industrial applications of isolates. On the other hand, through the microscopic observation of the isolates inoculated in the B4 medium, it was observed that the precipitation of CaCO3 at 28°C always takes place within and often near the bacterial colony. Meier et al. (2017) suggested that the formation of CaCO3 crystals outside the bacterial colony may be as- sociated with specific proteins. According to our knowl- edge, the consequence of such crystal formation in the biotechnological uses of these bacteria has not yet been determined. Experimental studies are necessary for more precise conclusions. The CaCO3 and MgCO3 weathering bacteria of the cave, just like bacteria with CaCO3 precipitation ability, have also a geomicrobiological contribution to the for- mation of cave structures. Besides, there is an increased potential for using these microorganisms in biotechno- logical, industrial and environmental fields, which in- clude soil desalination. Soil salinization is the increase of dissolved salts in the salt profile of the soil, and this prob- SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS ACTA CARSOLOGICA 52/1 – 2023 157 lem significantly reduces the crop yield (Parida & Das, 2005). Salt-affected soils contain calcium salts, including CaCO3, Ca(OH)2, CaSO4.2H2O, and CaCl2. Among the common salts of saline soils, CaCO3 and MgCO3 have the lowest solubility at 0.006 g/l and 0.039 g/l, respec- tively (Johnson, 2013). Salts with the least solubility have the potential to be used by microorganisms to improve salt-affected soils (Orhan et al., 2017). 37 % of our used isolates showed both CaCO3 and MgCO3 weathering ability (Table 3). These bacteria include species of Bacil- lus, Pseudomonas, Microbacterium and Serratia genera. These isolates may constitute good candidates for the im- provement of salt-affected soils and soil fertility. 83 % of the Bacillus pumilus identified in our study have dissolved the MgCO3 contained in the medium. These species can be good candidates for the dissolution of magnesium ions for soil remediation. On the other hand, Brevundimonus species identified in this study have shown high rate of MgCO3 dissolution ability. How- ever, since these species were low identified in this study, it is necessary to test their MgCO3 dissolution ability by multiplying the number of these species in order to de- termine whether they are good candidates. NRPS and PKS are two important genes encoding many biologically active secondary metabolites with dif- ferent biotechnological importance. These genes were detected in genera like Bacillus, Peannibacillus, and Pseudomonas from which important bioactive compounds have been secreted (Sukhanova et al., 2018). The NRPS genes in Bacillus and Pseudomonas have been reported to encode mainly for lipopeptide biosurfactant synthesize. In our study, B. pumilus, B. safensis, B. megaterium, B. zhangzhouensis, Pseudomonas species shown either through MT-F/MT-R or A3F/A7R have been successfully screened for the NRPS cluster. Lipopeptide families encoded by the NRPS from species of Bacillus including fengycin, pilpastatin, and surfactin have antimicrobial and immunosuppressive functions (Aleti et al., 2015; Sukhanova et al., 2018; Olishevska et al., 2019). In addition, xantholysin, massetolide A, and amphisin are lipopeptides, produced by species of Pseudomonas, showing antimicrobial activities against human pathogens and which can be used in biocontrol plant disease (Raaijmakers et al., 2010; Li et al., 2013; Ke- nawy et al., 2019). The PKS gene is detected in 5.9 % of our isolates, especially in Bacillus, Pseudomonas and Paenibacillus genera, by using the degenerate primer DegKS2F/DegK- S2R. The presence of this gene cluster in these genera has been observed in previous studies and it was related to the synthesis of a diversity of bioactive compounds. They include the antibiotics bacitracin and bacillaene synthe- sized by Bacillus genus. For those which are produced by Paenibacillus species, we can count paenimacrolidin and paenilamicin which have antimicrobial activities against a range of human and plant pathogen bacteria and fungi like MRSA, ampicillin-resistant Staphylococcus epidermi- dis, Bacillus strains and Saccharomayces cerevisiae (Oli- shevska et al., 2019). On other hand, bacteria of the ge- nus Pseudomonas are known for their wide synthesized secondary metabolites. Sukhonova et al. (2017) reported that species of Pseudomonas produce about 795 second- ary metabolites including 610 antibiotics and 185 sub- stances with diverse activities. Some compounds synthe- sized from the PKS gene clusters of Pseudomonas species include mupirocin, pyroles, benzaldehyde, quinolone, and moiramides which are known for their pharmaceu- tical importance. In our study, it has also been observed that the bio- active substances production potentials of the bacteria were not related to the isolation of these bacteria from the soil or surface but differ at the sampling points. From this point of view, increasing the sampling points in such studies will increase the chance of obtaining isolates with different characteristics. 5. CONCLUSION Bacteria used within the scope of this study have been screened for their role in caves and cave structure for- mations as well as for their potential in biotechnological and industrial areas. Results shown that our isolates have both CaCO3 precipitation and MgCO3 and CaCO3 dis- solution abilities. Based on the previous studies in this study field, these observed potentialities suggest that our isolates have a role in the formation and development of the Yarık Sinkhole. On the other hand, these two main activities (min- eral precipitation and dissolution) of our isolates provide ideas of the use of these isolates in diverse fields such as construction and bioremediation. In addition, NRPS and PKS gene clusters have been screened in these isolates and the observed results promise high potential uses of the Yarık Sinkhole’s isolates or their products for biotech- nological, industrial, and pharmaceutical needs. Lastly, one of the interesting observations noted in ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 52/1 – 2023158 this study is the crystal formation potential of non-ureo- lytic bacteria. These bacteria may be suggested to be used alternatively instead of the ureolytic ones due to the en- vironmental and social disadvantages that can be caused by the ureolytic bacteria. Besides, the crystal formation period, under laboratory conditions, as well as the mor- phologies were diversified even among the isolates of the same species. We suggest that all these observations should be considered in future studies for the selection of the best strains for biotechnological and industrial ap- plications. ACKNOWLEDGEMENTS This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University. Project num- ber: FBA-2018-29145. Our thanks are also directed to Burçin IREN for contribution. REFERENCES Ai, J., Guo, J., Li, Y., Zhong, X., Lv, Y., Li, J., Yang, A., 2022. The diversity of microbes and prediction of their function in karst caves under the influence of hu- man tourism activities- a case study of Zhijin Cave in Southwest China. 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Applied Microbiology and Biotechnology, 105: 8921-8936. https://doi.org/10.1007/s00253-021- 11658-4 ACTA CARSOLOGICA 52/1 – 2023 163 Y108 1S Bacillus pumilus /JQ773350.1/ 99 + - 2 + - + - Y109 1S Bacillus safensis MT501806.1/100 - - 1 + - + - Y110 1S Bacillus sp./ MG553999.1/100 + + 2 - - + - Y112 1S Morganella morganii / KY938128.1/99 - - 24 + - + - Y113 1S Bacillus pumilus / JQ773350.1/99 + - 17 + - + - Y116 1S Stenotrophomonas maltophilia / KF595070.1/99 + + 4 + - + - Y117 1S Micrococcus aloeverae / MT733991.1/99 + - 16 + - - - Y118 1S Bacillus safensis / MT733991.1/99 + + 21 + - + - Y119 1S Bacillus sp. / KU236475.1/99 + - 10 + - - - Y120 1S Bacillus sp. / MT598008.1/99 + - 15 + - + - Y121 1S Bacillus pumilus / HM744710.1/99 + - 14 + - + - Y122 1S Bacillus pumilus / LT906438.1/99 + - 2 + - + - Y123 1S Bacillus simplex / MN750767.1/100 + - 15 + - - - Y124 1S Bacillus pumilus / MN709186.1/95 + + 18 + ND ND ND Y125 1S Bacillus pumilus/ LT906438.1/100 + + 15 + - + - Y126 1S Bacillus megaterium / GU122960.1/100 + + 9 + - + - Y129 1S Bacillus pumilus / LT906438.1/100 - + 3 + - - - Y131 1S Bacillus pumilus / MN117687.1/99 + - 7 + - + - Y132 1S Bacillus safensis / MT642941.1/99 + - 18 + - + - Y133 1S Bacillus safensis / KY820904.1/99 + + 18 - - + - Y136 1S Bacillus pumilus / JQ773350.1/99 + + 8 + - - - Y138 1S Bacillus sp. / MG470694.1/99 + + 19 + - + - Y140 1Y Bacillus pumilus / CP058911.1/99 + + 9 + - - - Y141 1Y Bacillus safensis / MT642941.1/100 + - 18 + - + - APPENDIX Table. 2: Biomineralization, dissolution, and some genotypic characteristics of Yarık Sinkhole's isolates. Isolate code Sample area Bacterial division/Accession (Nearest relative)/ % Similarity MgCO3 dissolu- tion CaCO3 dissolu- tion Production of Crystal (Day) Crystal observed away from colonies PKS NRPS MT A3/A7 ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 52/1 – 2023164 Y143 1Y Bacillus safensis / KR054086.1/99 + - 15 + - + - Y146 1Y Bacillus safensis / MT501806.1/99 + + 1 - - - - Y147 1Y Micrococcus aloeverae / MN401101.1/99 - - 10 + - + - Y149 1Y Bacillus pumilus / KC182057.1/99 + - 19 + - - - Y150 1Y Serratia myotis / KJ739884.1/99 + + 18 - - - - Y152 1Y Microbacterium sp. / MG228460.1/99 + + 18 + - + - Y153 1Y Bacillus pumilus / LT906438.1/99 + + 8 + - - - Y154 1Y Micrococcus luteus / MH142592.1/98 - + 8 + ND ND ND Y157 1Y Bacillus pumilus / KC182057.1/99 + + 18 + - + - Y162 2Y Bacillus zhangzhouensis / MN826587.1/99 - + 18 + - + - Y165 2Y Bacillus pumilus / MT102721.1/100 - + 7 - - - - Y168 2Y ND + + 1 + + - - Y169 2Y Bacillus pumilus / CP054310.1/99 + + 15 + + - - Y171 2Y ND - + 5 + - - + Y172 2Y Brevundimonas vesicularis / MH283789.1/99 + + 15 + - - - Y173 2Y Brevundimonas vesicularis/ KJ540983.1/99 + - 18 + - + - Y175 2Y Bacillus zhangzhouensis / MT538321.1/100 - - 21 + - + - Y177 2Y Bacillus sp. / MK491013.1/100 - + 3 + - - - Y178 2Y Sphingomonas mucosissima / MK415046.1/99 - - 2 + - - - Y179 1S Bacillus sp. / MF373581.1/100 - - 18 - - + - Y181 1Y Bacillus pumilus / CP016784.1/99 + + 2 - - + - Y184 2Y Pseudomonas sp. / MT626781.1/99 - - 8 + + - + Y186 2Y Brevundimonas vesicularis/ MN932333.1/100 + + 18 + - - + Y187 2Y Brevundimonas vesicularis / KJ540983.1/99 + + 18 + - - - Y188 2Y Bacillus pumilus / LT906438.1/99 + + 18 - - - - Isolate code Sample area Bacterial division/Accession (Nearest relative)/ % Similarity MgCO3 dissolu- tion CaCO3 dissolu- tion Production of Crystal (Day) Crystal observed away from colonies PKS NRPS MT A3/A7 SCREENING OF BACTERIA IN YARIK SINKHOLE, ANTALYA, TURKEY FOR CARBONATE DISSOLUTION, BIOMINERALIZATION AND BIOTECHNOLOGICAL POTENTIALS ACTA CARSOLOGICA 52/1 – 2023 165 ELIF ÖZLEM ARSLAN- AYDOĞDU, YAĞMUR AVCI, NAHDHOIT AHAMADA RACHID, BATU ÇOLAK & NIHAL DOĞRUÖZ-GÜNGÖR Y189 2Y Bacillus zhangzhouensis / MN826587.1/100 - + 18 - - + - Y193 2Y Bacillus sp. / MG470705.1/99 - - 1 + - - - Y196 2S Bacillus pumilus / KC182057.1/99 + + 18 + - + - Y199 2S ND - + 9 + - + - Y200 2S Bacillus pumilus / CP054310.1/99 + - 18 + - + - Y201 2S Bacillus safensis / MT642941.1/100 - + 15 + - + - Y202 2S Bacillus sp. / MT808965.1/98 + - 17 + - - - Y205 2S Paenibacillus apiarius / FR877661.1/100 - + 21 + + - - Y208 2S Staphylococcus epidermidis/ MT445363.1/99 - + 1 + - - - Y211 2S Bacillus pumilus / MN181331.1/100 - + 18 + - - - Y212 2S Bacillus zhangzhouensis / MN826587.1/99 - - 1 + - - - Y213 2S Bacillus safensis / MT733991.1/99 - - 1 + - + - Y218 1Y Bacillus safensis / MT501806.1/100 - - 14 + - - - Y222 2S Bacillus safensis / MT501806.1/100 - - 8 + - + + Y227 1Y ND - + 3 + - - - Y228 1Y Bacillus pumilus / LT906438.1/100 + + 18 - - + - Y229 1Y Bacillus safensis / MT642941.1/100 + + 21 - - - - Y232 1Y Bacillus pumilus / LT906438.1/99 + - 18 + ND ND ND Y271 2S Bacillus safensis / MH321592.1/99 + + 3 + - + - Y276 2S Bacillus sp. / LC488930.1/99 - + 18 + - + - Y281 2S Bacillus pumilus / MN581193.1/99 - + 17 + - - - S: soil; Y: surface; 1: -80 m, 2:-300 m, ND : Non determined Isolate code Sample area Bacterial division/Accession (Nearest relative)/ % Similarity MgCO3 dissolu- tion CaCO3 dissolu- tion Production of Crystal (Day) Crystal observed away from colonies PKS NRPS MT A3/A7 ACTA CARSOLOGICA 52/1 – 2023166