THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY STUKTURA MIKROBNE ZDRUŽBE V JAMI DUPNISA, KIRKLARELI, TURČIJA Nihal DOĞRUÖZ-GÜNGÖR 1* Abstract UDC 579:551.44(560) Nihal Doğruöz-Güngör: The microbial community structure of the Dupnisa cave in Kırklareli, Turkey Cave ecosystems count to be extreme environments due to their stable temperature, darkness, high humidity rates, and limited organic materials. In this context, these ecosystems represent invaluable laboratories for microbiological studies. Although there are common features between the microor- ganism groups obtained from the culture-based microbiologi- cal studies conducted in the caves and the groups highlighted through molecular methods, the microorganism groups deter- mined through this last method are richer. The detected mi- croorganisms are variable depending on the characteristics of each cave. The aim of this study is to determine the microbial diversity in samples taken from 5 different regions (including regions visited by tourists) of Dupnisa Cave and to reveal the differences between these regions. This is the first microbiologi - cal study running in cave sediments of Dupnisa Cave System situated in the north-western of Turkey. In this study, using the Illumina MiSeq next-generation sequencing approach for analyses of Dupnisa Cave samples, 14 phyla and 298 genera as well as 2 phyla and 20 genera can be attributed to bacterial and archaea OTUs, respectively. Moreover, the bacterial commu- nity is dominated by the phyla Proteobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Firmicutes, Nitrospirae, Chloroflexi and Acidobacteria distributed with 1 % and above. Archaeal community is represented by Thaumarchaeota and Euryarchaeota phyla. Proteobacteria is the most dominant bacterial phylum and Thaumarchaeota dominates the archaeal phyla. The highest number of types of bacteria according to Chao 1 richness estimation index were found at point AF (cave entrance / sediment), and that of types of archaea were found at point F2 (touristic area 2 / cave sediment). F2 was determined as the sampling point with the highest diversity of archaeal and bacterial genera according to Shannon-Wiener diversity index. Key words: cave microbiology, bacterial diversity, archaeal di- versity, next generation metagenomic sequencing. Izvleček UDK 579:551.44(560) Nihal Doğruöz-Güngör: Struktura mikrobne združbe v jami Dupnisa, Kırklareli, Turčija Stabilna temperatura, tema, velika zračna vlaga in omejena količina organske snovi so dejavniki, ki jamski ekosistem opredelijo kot ekstremno okolje in mu dajejo velik pomen v mikrobioloških študijah. Čeprav so bili izsledki študij z gojit- venimi metodami v jamah podobni izsledkom, pridobljenim z molekularnimi metodami, so zadnjenavedene omogočile določitev pestrejšega nabora mikroorganizmov. Spremenlji- vost mikroorganizmov se razlikuje glede na značilnosti posa- mezne jame. Cilj te študije je opredeliti raznovrstnost mikrobov v vzorcih iz petih predelov (vključno s turističnim predelom) jame Dupnisa in odkriti razlike med temi predeli. To je prva mikrobiološka študija jamskih sedimentov iz jamskega sistema Dupnisa na SZ T určije. S sekvenciranjem Illumina Miseq nasled- nje generacije je bilo 14 debel in 298 rodov pripisanih bakteri- jskim OTU, 2 debli in 20 rodov pa arhejskim OTU. V bakteri- jski združbi prevladujejo debla Proteobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Firmicutes, Nitrospirae, Chloroflexi in Acidobacteria, združbo arhej pa zastopata debli Thaumarchaeota in Euryarchaeota. Med bakterijami je vodilno deblo Proteobacteria, pri arhejah pa Thaumarchaeota. Indeks pestrosti Chao1 kaže na največjo pestrost bakterij na vzorčnem mestu AF (vhod v jamo/sediment), največjo pestrost arhej pa na vzorčnem mestu F2 (turistični del 2/jamski sediment). Glede na Shannon-Wienerjev indeks pestrosti je bila največja razno- likost rodov arhej in bakterij na vzorčnem mestu F2. Ključne besede: jamska mikrobiologija, raznovrstnost bakterij, raznovrstnost arhej, sekvenciranje naslednje generacije. 1 Istanbul University, Faculty of Science, Department of Biology, 34134, Istanbul, Turkey, e-mail: ndogruoz@istanbul.edu.tr * Corresponding author Received/Prejeto: 06.04.2020 DOI: 10.3986/ac.v49i2-3.8575 COBISS: 1.01 ACTA CARSOLOGICA 49/2-3, 281-295, POSTOJNA 2020 INTRODUCTION Cave habitats are defined as dark underground gaps with openings to the surface and characterized by lim- ited amount of organic matter as well as large mineral surface areas (Palmer 1991; Northup & Lavoie 2001). Therefore, they are considered as extreme environ- ments for the inhabitation of certain microorganisms (Lee et al. 2012). Organic matter entrance, essential for sustaining life in caves, is facilitated via dripping water, underground rivers, visitors and animals, particularly bats (Groth et al. 1999; Groth & Saiz-Jimenez 1999; Barton 2006). However, in some caves, lithotrophic bacteria play a primary role in supporting the growth of heterotrophic bacteria (Chen et al. 2009; Porter et al. 2009). The uniqueness of caves is likely to account for a rich genetic potential and microbial diversity. However, the cave temperature, CO 2 concentration and amount of water vapor increase in proportion to the number of tourists entering the cave and may cause changes in microclimatic conditions and food chain (Hoyos et al. 1998). Microbial diversity will also be affected as a re- sult of these changes. High organic material input may lead to the disappearance of cave microorganisms by making the cave nonnative microorganisms more com- petitive than the cave inhabitants (Bastian & Alabou- vette 2009; Chelius et al. 2009; Mulec & Kosi 2009; Fong 2011). Culture methods were till recently used in most cave microbiology studies. However, as is well-known, culture-based methods being often inadequate in fully assessing the microbial diversity in a specific environ- ment. Increasing use of molecular methods has led to a better understanding of cave diversity and cave eco- system characteristics. The next generation sequenc- ing technology enables much more microorganisms to be detected with high sensitivity at the DNA level and identifies non-cultured microorganisms. Such tech- niques shed light on current studies about caves in the field of biology and biotechnology. 40 % of the Turkey is covered with karstic carbon- ate and sulfate rocks. Because of that, this country has standing as the richest karst areas in European coun- tries. Although there is still a significant number of un- discovered caves in Turkey, it is estimated based on re- search made in karstic areas that, around 35.000-40.000 caves may well be found spread all over the country (General Directorate of Mineral Research and Explora- tions 2020). Our study aims to determine and compare the microbial diversity of the Dupnisa Cave regions, open to tourism as well as those which are closed; therefore, the community structure of bacteria and archaea in samples collected from the Kuru Cave and Sulu Cave regions of the cave is determined using the next genera- tion Illumina MiSeq sequencing approach. CAVE DESCRIPTION Dupnisa Cave, located in Kırklareli-Sarpdere Village, the north-west of Turkey, is a horizontal cave system of 2720 m long (Paksuz 2017). This system comprises three interconnected caves, so-called Kuru Cave (41 ° 50’20”N; 27 ° 33’26”E), Sulu Cave (41 ° 50’29”N; 27 ° 33’25”E) and Kız Cave (41 ° 50’07”N; 27 ° 33’28”E), which hydrologically contains fossil zones, semi-active and active zones (Fig. 1) (Nazik et al. 1998; Paksuz et al. 2007; Paksuz 2017). Dupnisa Cave System was opened to visit in 2003. Since then, it has been visited by nearly 35,000 visitors each year. First 200 m of Sulu Cave and 230 m of Kuru Cave contain walkways and lighting used by tourists. However, Kız Cave is fully closed to visitors. On the other hand, for the protection of hibernating bats and their secure repro- duction, the Sulu Cave is closed to visitors only between November 15 th and May 15 th (Paksuz 2017; Nazik et al. 1998). Dupnisa Cave has been evolved due to breakage of Pliocene relief system by quaternary streams and it pos- sesses a polycyclic growth character (Nazik et al. 1998). Kuru and Kız Caves at the top have completely become fossilized. Sulu Cave, at the bottom, is the longest one (1977 m) and has an active structure in which cave formation is still progressing. It has lakes up to 2 m in depth and a large underground stream with continuous flow. Kızılcık Stream, originating from mountains at the south, comes back to the surface after passing through Sulu Cave (Nazik et al. 1998). The most of organic ma- terial entering the Dupnisa Cave is provided by the guanos of 18 bat species living in it (Paksuz 2017) and the river flowing through the cave. In addition, artificial lighting at the entrance and touristic part of Dupnisa Cave supports the development of phototrophic micro- organisms. NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 282 THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY MATERIALS AND METHODS SAMPLING SITE A total of 5 cave sediment samples were taken from Sulu and Kuru Caves which are parts of the Dupnisa Cave Sys- tem in November 2015. Two of these samples were col- lected from a tourist area of Kuru Cave (F1 cave sediment sample; F2 cave sediment sample), while the other three ones were from Sulu Cave. One of the samples collect- ed from the Sulu Cave was from the entrance (AF cave sediment sample) which is a touristic area (Fig. 1). The two other ones were collected from the dark zone, a non- tourist area (A1 cave sediment sample; A2 cave sediment sample) (Fig. 1). PHYSICOCHEMICAL PROPERTIES OF THE ENVIRONMENT The physicochemical properties of the studied environ- ment have been determined through the water stream from where A1 and AF samples have been collected. Two aliquots of water collected from A1 and AF areas in the cave were measured in situ in terms of temperature, pH, dissolved oxygen, conductivity and total dissolved solids (TDS) using a portable multiparameter tool (a Hach Lange HQ40D multimeter). Air temperatures and humidity at the five investigation sites were measured by a portable Temperature/Humidity Meter (TFA 31.1O28). DNA ISOLATION The DNA was extracted from 0.3 g (wet weight) of cave sediment samples by using FastDNA SPIN Kit for Soil (MP Biomedical, Santa Ana, CA, USA) following the manufacturer’s instructions. 16S RRNA METAGENOMIC SEQUENCING LIBRARY PREPARATION AND SEQUENCING Isolated genomic DNAs were used for two-step PCRs in order to prepare 16S rRNA gene amplicon librar- ies. Each sample was subjected for two library prepara- tions, archaeal and bacterial. Archaeal V4-V5 (Arch519F (5’-CAGCCGCCGCGGTAA-3’) / Arch 915R (5’-GT- GCTCCCCCGCCAATTCCT-3’) regions and bacte- rial V3-V4 (341F: 5’-CCTACGGGNGGCWGCAG-3’ / 805R: 5’- GACTACHVGGGTATCTAATCC-3) regions of 16S rRNA gene were targeted for amplicon sequenc- ing. The protocol includes the primer pair sequences for the targeted regions of the 16S rRNA. It also includes overhang adapter sequences appended to the primer pair sequences for the compatibility with the Illumina index and sequencing adapters. By using BiospeedyTM Proof Reading DNA Polymerase, 2x Reaction Mix (Bioeksen Ltd Co., Turkey) and 200 nm of each primer, the first PCR was realized on Biorad CFX Connect Instrument (Bio-Rad Laboratories, U.S.A.) under this program: 95 °C for 3 minutes, 25 cycles of (95 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 30 seconds); 72 °C for 5 minutes. To verify the size (~550 bp), the PCR product was run on an agarose gel, then purified by using Bio- speedyTM PCR Product Purification Kit (Bioeksen Ltd. Co., Turkey).  The dual indices and Illumina sequencing adapters were attached to the purified first PCR products via the second PCR, which was run using the Nextera XT Index Kit (Illumina Inc., USA) with the following program: 95 Fig. 1: The Dupnisa Cave System located in Turkey and sampling points (Adapted from Nazik et al. 1998). ACTA CARSOLOGICA 49/2-3 – 2020 283 °C for 3 min, 8 cycles of (95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s) and 72 °C for 5 min. The PCR products were purified using a Biospeedy® PCR Product Purifica- tion Kit. The final library was run on a Bioanalyzer DNA 1000 chip to verify the size (~630 bp). The final library was diluted by using 10 mM Tris (pH 8.5) to 4 nM and the 5 mL aliquots were mixed for pooling the libraries. In preparation for the cluster generation and sequencing, pooled libraries were denatured with NaOH, diluted with hybridization buffer (HT1), and then heat denatured be- fore the MiSeq sequencing. Illumina MiSeq v3 reagent kits were used for the runs. BIOINFORMATIC ANALYSIS Following the Mothur Version 1.36.1, the raw sequence data (concatenated forward and reverse sequence reads) were cleaned, reduced and analysed. First, the barcode and the primer sequences were trimmed in order to iden- tify unique sequences. Then, using BLASTn algorithm (Pruesse et al. 2007) this unique sequences were aligned to the SILVA rRNA database sequences. Bacterial and archaeal 16S rRNA gene amplicon data were analysed separately. The extending at the ends was putted out by filtering the sequences, then the redundancy check was carried out. The sequences were pre-clustered in order to further de-nosing. By utilizing the implanted code UCHIME (Edgar et al. 2011) the chimeras were exclud- ed. The sequences were classified by using Bayesian clas- sifier implanted in Mothur. The reference and taxonomy files were chosen from the SILV A database (Pruesse et al. 2007). After Operational Taxonomic Unit (OTU) picking and their taxonomic assignment using the SILVA rDNA database, the OTUs were binned into phylotypes. Addi- tionally, the microbial diversity was analysed within the sample by calculating Shannon and Chao1. Chao was used to determine community richness, and Shannon in- dex was used to determine community diversity. SEQUENCE ACCESSION NUMBERS The raw sequencing data generated in this study were deposited into the NCBI database under the acces- sion numbers SAMN08142743, SAMN08142744, SAMN08142745, SAMN08142746 and SAMN08142747. RESULTS PHYSICOCHEMICAL ANALYSIS The cave sediment samples taken from five designated sites of the Dupnisa Cave were analysed. The air tem- peratures and humidity values of the five investigation sites are shown in Tab. 1. In addition, physicochemical analysis of the waters where the cave sediment samples were taken are shown in Tab. 2. BACTERIAL TAXONOMY AND DISTRIBUTION The bacterial diversity of the Dupnisa Cave has been in- vestigated at five different sampling locations. All the se- quence reads were assigned into 1109 OTUs (based on 97 % cutoff) at the genus level of the taxonomy. The bacterial OTUs can be assigned into 14 phyla, 64 orders, 130 fami- lies and 298 genera. The bacterial diversity in the sam- NIHAL DOĞRUÖZ-GÜNGÖR Tab. 1: Sampling sites, air temperature and humidity. Sampling Location Cave Abbreviation Parts Air temperature ˚C Air humidity % Water spring Sulu A1 Active 13.1 78 % Last gallery Sulu A2 Active 12.6 95 % Touristic area 1 Kuru F1 Fossil 15.2 81 % Touristic area 2 Kuru F2 Fossil 14.9 79 % Cave entrance Sulu AF Active 10.6 71 % Tab. 2: Physicochemical parameters from water samples. Water Samples Location Water temperature ˚C pH Conductivity (µs/cm) TDS (mg/L) O2 (mg/L) Water spring (A1) 12.2 8.7 290 178 10.2 Cave entrance (AF) 9.8 8.5 255 156 10.3 ACTA CARSOLOGICA 49/2-3 – 2020 284 ples taken from the active and fossil parts of the Dupnisa Cave System was found to be quite different. The highest diversity was observed on F2 and AF samples (Fig. 2). Only eight phyla, Actinobacteria, Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, Nitrospirae, Chloroflexi, and Gemmatimonadetes, containing more than 98 % of the total sequence read, show mean abun- dance more than 1 % in at least one sample holding more than 98 % of the total sequence reads. For Actinobacteria and Proteobacteria, the abundance rate in all sampling locations is above 1 %. The most dominant phylum both in the active and fossil region of cave is Proteobacteria except in A2 area (Fig. 2). The most predominant phylum in A2 was observed to be Bacteroidetes (64.2 %). Yet, in the other sampling regions, Bacteroidetes remain below 1 %. Besides, Chloroflexi and Nitrospirae phyla remain THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY Fig. 2: Relative abun- dance of the bacterial community at the phy- lum (A) and genus (B) level. Samples sites; A1 (Water spring), A2 (Last gallery), F1 (Touristic area 1), F2 (Touristic area 2), AF (Cave en- trance). A B ACTA CARSOLOGICA 49/2-3 – 2020 285 above 1 % abundance rate at F2 and AF sampling points, and Gemmatimonadetes is above 1 % only at F2 sam- pling point. At the class level, Alpha-Proteobacteria and Acti- nobacteria were present at all sample areas. Alpha-Pro- teobacteria was the dominant class of phylum Proteo- bacteria, ranging from 10.6-83.2 % within the samples, compared to other Proteobacteria classes; Beta-Proteo- bacteria (0.04-11 %), Gamma-Proteobacteria (0.03-18 %) and Delta-Proteobacteria (0.02-1.5 %). Except for the second active area (A2), Alpha-Proteobacteria was found to be the most dominant class in both fossil and active areas. Among the Alpha-Proteobacteria, Nordella (35.5 % at AF), Ochrobactrum (33.2 % at F1) and Methylobacte- rium (41 % at A1 and 49.3 % at F1) are observed as the most predominant genera. The bacteria which are found more abundant at F2 is Dongia (5.4 %) which belongs to Alpha-Proteobacteria family. It is also observed that Gamma-Proteobacteria is more predominated in the fos- sil locations at F1 (12 %) and at F2 (18 %). While the Acinetobacter genus of Gamma-Proteobacteria is found predominantly at F1, the abundance of Dokdonella (4 %) and Beggiatoa (4.2 %) genera are highly expected at F2. The Beta-Proteobacteria is found in its highest rate at A2 and Massilia (8.8 %) is the most predominant genus ob- served. Actinobacteria class is found in range of 2.9-40 % with the most detected genera were Propiniobacterium (39.8 %) at A1 and Arthrobacter (6.8 %) at A2. Flavobac- terium genus (63.8 %), which belongs to Flavobacteriia class, is revealed to be the most predominating genus at A2. In this study, a total of 130 families were obtained. 19 families Rhodocyclaceae, Hyphomicrobiaceae, Propi- onibacteriaceae, Rhizobiaceae, Bacillaceae, Flavobacte- riaceae, Brucellaceae, Beijerinckiaceae, Methylobacteria- ceae, etc., were commonly shared by all samples. There were 20 rare families that only appeared in one sample, such as Parvularculaceae, Legionellaceae, Dehalococ- coidia, Paenibacillaceae, Streptosporangiaceae, Sangui- bacteraceae, Chloroflexi, etc., that were found (<1 %) in samples except Corynebacterineae (>1 %). The most genera were determined in AF point (208 genera) while the fewest were found in A1 point (66 gen- era) (Tab. 3). Shannon diversity index (to measure diver- sity by taking into account the number of genera/species as well as their abundance) for bacteria shows that, F2 had the highest diversity (Shannon = 5.61), which was much larger than the diversity of A1 (Shannon = 1.20) among the five communities (Tab. 4). Chao1 index (esti- mation of total genus/species richness) for bacteria, indi- cates that AF had the highest bacterial richness (Chao = 768), while A1 had the lowest (Chao = 306). Tab. 3: Number of bacterial and archaeal genus at the sample points. A1 A2 F1 F2 AF Number of genus (All) B 66 190 74 188 208 Number of genus (>1 %) B 4 9 5 29 16 Number of genus (All) A 9 9 9 19 12 Number of genus (>1 %) A 9 6 9 7 9 B: Bacteria; A: Archaea Tab. 4. Characteristics of cave bacteria richness and diversity indi- ces among different sample points of Dupnisa Cave. A1 F1 A2 F2 AF Shannon-Wiener Diversity Index (Bacteria) 1.20 1.34 3.68 5.61 4.97 Chao1 Richness Estimation (Bacteria) 306 709 721 681 768 ARCHAEAL TAXONOMY AND DISTRIBUTION A total of 118 OTUs (based on 97 % cutoff) comprising 2 archaeal phyla were detected in this study. Abundance rate of Euryarchaeota in the samples taken from the ac- tive and fossil parts of the Dupnisa Cave System was found between 8.2 %–16 %, whereas that of Thaumar- chaeota was between 84 %–91.8 %. The archaeal OTUs can be assigned into 11 orders, 14 families and 20 genera (Fig. 3). Thaumarchaeota class from Thaumarchaeota phy- lum and Methanomicrobia class from Euryarchaeota phylum were determined above 1 % at all sampling points. However, Methanobacteria and Methanococci class members exceeded 1 % abundance rate only at A1, F1, and F2 sampling points. Nitrososphaeraceae, Cenar- chaeaceae, Nitrosopumilaceae are the families which has 5 % over all the samples taken from the active and fos- sil parts of the Dupnisa Cave System. Nitrososphaera- ceae was determined as the most dominating group in all samples (58.5-68.8 %), except at the AF point. The highest number of archaeal genera was determined at F2 (Tab. 3). Abundances of Candidatus Nitrososphaera and Nitrososphaera archaeal genera at A1, A2, F1 and F2 points were between 30.5-43.8 % and 25-28.7 %, respec- tively, whereas, they were 10.9 % and 2.3 % respectively at AF point. The most dominating genus at AF was Ce- narchaeum (49 %). Besides, Thermococcus (4.5 %), Ther- moplasma (3.3 %) and Thermogymnomonas (3.2 %) gen- era were only determined at AF samples. Abundance of Nitrosopumilus at A2 (4.4 %) was much lower than those measured at the other studying sites (12.5-19.8 %). On the other hand, Candidatus Nitrosotalea and Methano- saeta were the most abundant at the A2 site with 15.6 % NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 286 and 15.4 %, respectively. However, Candidatus Nitroso- talea was not detected at the other points, while Metha- nosaeta was observed between 1.5-3.1 % at these points. Tab. 5: Characteristics of cave archaea richness and diversity indi- ces among different sample points of Dupnisa Cave. A1 F1 A2 F2 AF Shannon-Wiener Diversity Index (Archaea) 2.64 2.20 2.49 2.66 2.14 Chao1 Richness Estimation (Archaea) 106 43 34 111 57 The most genera were determined in F2 point (19 genera). Chao1 index (estimation of total genus/species richness) for archaea indicates that the F2 sample had also the highest richness (Chao = 111), followed by A1 (Chao = 106), and the lowest one (Chao = 34) was ob- served at A2. Shannon diversity index (to measure di- versity by taking into account the number of genera/spe- cies as well as their abundance) for archaea shows that, F2 had the highest diversity (Shannon = 2.66) followed closely by A1 whereas AF had the lowest one (Shannon = 2.14) (Tab. 5). THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY A B Fig. 3: Relative abun- dance of the archaeal community at the fam- ily (A) and genus (B) level. Samples sites; A1 (Water spring), A2 (Last gallery), F1(Touristic area 1), F2 (Touristic area 2), AF (Cave entrance). ACTA CARSOLOGICA 49/2-3 – 2020 287 DISCUSSION In this study we investigated the microbiome of the Dup- nisa Cave, Kırklareli, Turkey by Illumina. Paksuz et al. (2007) stated that the humidity in the Dupnisa Cave Sys- tem varied between 57 % and 100 % and found that the Sulu Cave humidity was higher than the Kuru Cave one. In addition, Paksuz et al. (2007) reported that in Sulu Cave, the average temperature in winter is 7.4 °C and in summer 10.7 °C while in Kuru Cave in winter it is 11.4 °C and in summer it is 16.3 °C. According the physicochem- ical characteristics of caves in general, Dupnisa Cave ecosystem is a typical example of most cave systems with high relative humidity and limited temperature changes due to its connection with the surface (Paksuz 2017). It was determined that the pH, O 2 , conductivity and TDS values of the waters dripping over the cave were very similar at points A1 and AF. Conductivity is an indirect measurement of salinity and total dissolved solids (TDS) content (Al Dahaan et al. 2016). These details highlight the similarity in chemical environment existing within the cave areas. Culture-based techniques are known to release only 0.1-1 % of the total microorganisms (Torsvik & Øvreas 2002). With the development of molecular methods, mi- crobial diversity studies have also gained a new breath. Next Generation Sequencing (NGS) is one of these meth- ods. Illumina, which is one of the NGS platforms used in our study, has important advantages such as having relatively cheap price per base and high sorting depth despite short length readings (Van Dijk et al. 2014). In culture-based studies, it was determined that cave iso- lated bacteria belong mostly to Proteobacteria, Actino- bacteria, Bacteroidetes and Firmucutes groups (Barton & Jurado 2007; Zhou et al. 2007; Jurado et al. 2010; Barton 2015), while in non-culture based studies, groups such as Acidobacteria, Planctomycetes, Chloroflexi, Bacte- roidetes, Gemmatimonadetes, Firmicutes, Nitrospirae and Actinobacteria have been identified (Chelius & Moore 2004; Zhou et al. 2007; Lee et al. 2012; Oliveira et al. 2017; Lavoie et al. 2017). In our study, Proteobacte- ria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Firmicutes, Nitrospirae, Chloroflexi and Acidobacteria phyla (1 % and above) were also observed. De Mandal et al. (2015) and De Leon et al. (2018) have also identified Proteobacteria, Chloroflexi, Actinobacteria, Bacteroide- tes, Firmucutes and Planctomycetes phyla in bat guanos. Proteobacteria phylum, having a key role in biogeo- chemical cycles, and being abundant in samples from cave sediment, soil, dripping water and cave surface is a cosmopolitan bacterial group (Zhou et al. 2007; Tomc- zyk-Zak & Zielenkiewicz 2016). It was also found to be the most common and abundant phylum in cave sedi- ment samples taken from Dupnisa Cave. It is estimated that this phylum members involved in many biochemical processes in oligotrophic environments, may be primary producers in cave communities (Tomczyk-Zak & Zielen- kiewicz 2016). The composition and proportions of Pro- teobacteria population were found to be high in caves having interactions with humans, especially in those open to tourism (Northup et al. 2003; Ikner et al. 2007; Bastian & Alabouvette 2009; Shapiro & Pringle 2010). The most dominant class in the Proteobacteria in Dupni- sa Cave is Alpha-Proteobacteria, except at the point A2. Methylobacterium, the most dominant genus in A1 and F1, which can use a wide range of carbon substrates, per- form Type I formaldehyde assimilation and can be found in various media, have been determined in caves in previ- ous studies (Barton et al. 2004; Ilkner 2007). In addition, the second dominant Proteobacteria group identified in our study is Gamma-Proteobacteria similar to the results observed in some previous study (Chen et al. 2009; Jones et al. 2010). It was suggested that Acinetobacter, which was found to have the highest rate (12 %) at F1, mobi- lized inorganic phosphate and could provide us impor- tant information about feeding in nutrient-limited cave ecosystems (Tomczyk-Zak & Zielenkiewicz 2016). The Gamma-Proteobacteria class is known to contain various metabolically and ecologically chemolithotrophs which can use various inorganic molecules as the electron source (Tomczyk-Zak & Zielenkiewicz 2016). Thereby, they might play a vital role in sustaining diverse groups of other microorganisms in this ecosystem. Barton et al. (2007) reported, similarly with our result, that the most abundant genus in the Carlsbad Cave (USA) is Massilia belonging to the Beta-Proteobacteria group. Members of this genus produce acid by using carbon-carbohydrates and organic compounds as carbon and energy sources (Kersters 2006). This acid, which is corrosive to calcite, can play a role in the structural change of caves. Actinobacteria, the second dominant group in the Dupnisa Cave, is one of the most dominant and wide- spread phyla in the caves. Studies have shown that this phylum is found in cave walls, soil, sediment and speleo- them surfaces and it has been suggested that it can play an important role in the formation of biomineralization and cave structures in the cave ecosystem (Pašić et al. 2010; Cuezva et al. 2012; Ortiz et al. 2013; Tomczyk-Zak & Zielenkiewicz 2016). Arthrobacter genera, which was found in the percentage of 6.8 % at point A2, is interpret- ed to be responsible from the mineralization of calcite by the studies carried out (Rusznyák et al. 2012; Baskar et al. 2018). According to research conducted by the Human NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 288 Microbiome Project Consortium (2012), we considered the following genera might be related of human origin: Lactobacillus, Propionibacterium, Streptococcus, Bacteroi- des, Corynebacterium, Staphylococcus, Moraxella, Hae- mophilus, Prevotella and Veillonella. Propionibacterium has been observed at high rate in only the sample point A1 which is not open for tourism and thought to have no human impacts. Although the studies have been asso- ciated with human contamination (contamination dur- ing sampling or at the areas with human interaction), no other bacteria related with human contamination such as Streptococcus and Staphylococcus were identified. In addi- tion, the river flowing through Dupnisa Cave can reduce both human influences and carry organic matter from the outside into the cave. Further studies on this subject will have an important place in revealing this point. The effect of most microbial communities detected in the cave has not been revealed yet on the ecology of the cave. It has been reported that Acidobacteria, Gem- matimonadetes, Nitrospirae, Chloroflexi phyla, which constitute a small percentage of the microbial commu- nity of the Dupnisa Cave, are important members of the cave ecosystems (Schabereiter-Gurtner et al. 2002; Pašić et al. 2010; Rusznyak et al. 2012; Hathaway et al. 2014; Wiseschart et al. 2018). Some members of Acidobac- teria, which is found at small percentages in the caves, has methylotrophic growth ability and has an ecological advantage in an oligotrophic habitat with low organic matter input (Kalyuzhnaya et al. 2008). Chloroflexi phy- lum was detected over 1 % in the samples taken from the cave entrance (1.5 % AF) and tourist section (7.5 % F2) of Dupnisa Cave. Although members of this group are known to play a role in bioremediation, their ecological role in the cave system has not been identified yet (Wu et al. 2019; Yan et al. 2019). Like Acidobacteria and Chlo- roflexi phyla, information about the potential metabolic functions of Bacteroidetes in caves is limited (Schabereit- er-Gurtner et al. 2002; Chelius & Moore 2004; Macalady et al. 2006; Ikner et al. 2007; Rusznyák et al. 2012). As in Carlsbad Cavern and Krubera-Voronja Abkhazia, Geor- gia caves, the genus Flavobacterium was identified at the point A2 of Dupnisa Cave (Griffin et al. 2014; Kieraite- Aleksandrova et al. 2015). It has been reported to be ac- tive in the formation of ferromanganese deposits with their capacity to oxidize Mn (II) (Carmichael et al. 2013). Nitrospira genus which is a member of Nitrospirae and which was found in the percentage of 3.8 and 4.1 % at F2 and AF points respectively, is nitrite oxidizing bacte- ria which can convert ammonia (NH 3 ) to nitrite (NO 2 ). Members of this bacteria have been detected in many caves such as Pajsarjeva Jama Cave, Slovenia, Oylat Cave, Turkey and Tito Bustillo Cave, Spain (Schabereiter-Gurt- ner et al. 2002; Pašić et al. 2010; Gulecal-Pektas 2016). They support primary production by oxidizing the nitrite in caves (Ortiz et al. 2013). It is evaluated that archaeal communities have important roles in cave ecosystems and the exis- tence of Euryarchaeota, Crenarchaeota, Korarchaeota, Thaumarchaeota and some candidate phylum was ob- served in caves. However, the information obtained from the studies is still limited (Engel 2010; Carmichael et al. 2013; Jones et al. 2014; Kieraite-Aleksandrova et al. 2015). Only the phylum Thaumarchaeota and Euryarchaeota were identified in the Dupnisa Cave. The newly discov- ered Thaumarchaeota is known to be a chemoautotro- phic ammonia oxidizer and is remarkable for its ability to developing in oligotrophic environments such as caves (Martens-Habbena et al. 2009; Pester et al. 2011; Ortiz et al. 2014; Kimble 2017; Zhao et al. 2017). In caves, ammo- nia from organic matter mineralization and / or guano deposits may be an energy source for chemolithotrophic organisms (Hathaway et al. 2014). In fact, the archaeal community analysis of bat guano sampled in the Domica Cave showed that the archaeal ammonia oxidizers were dominant as it is in Dupnisa Cave samples (Chronako- va et al. 2009). Furthermore, Thaumarchaeota (over 80 %) was identified as the dominant archaeal phylum in the Dupnisa Cave, like in Kartchner Caverns (Arizona, USA) and Weebubbie Cave (Arizona, USA) (Bates et al. 2011; Tetu et al. 2013). Members of Thaumarchaeota are the dominant players in global nitrification (Stahl &De la Torre 2012; Ortiz et al. 2014; Zhao et al. 2017). They could be important since the nitrogen cycle provides nu- trients and energy for microorganisms in oligotrophic environments such as caves (Tetu et al. 2013; Ortiz et al. 2014; Kimble 2017; Zhao et al. 2017). Ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) are responsible for the micro- bial oxidation of ammonia (Bartossek et al. 2012; Stahl and De la Torre 2012; Ortiz et al. 2014). In our study, Nitrosopumilus, Nitrososphaera, Candidatus Nitrosos- phaera and Cenarchaeum were predominantly detected at all sampling points. It was reported that Nitrosopumi- lus is an ammonia oxidizing marine archaeon (mem- ber of group I) and Nitrososphaera is frequently found in terrestrial environments (Tetu et al. 2013; Ortiz et al. 2014). The results of studies held by Anda et al. (2017) in Molnár János Cave, Hungary showed an archaea di- versity like those of the Dupnisa Cave. Only members of Thaumarchaeota and Euryarchaeota phyla were de- tected in this cave and it was reported that the percent- age of Thaumarchaeota phylum was very high. The most abundant genus of Thaumarchaeota phylum found in Molnár János Cave are Candidatus Nitrososphaera and Nitrososphaera. Similarly, in Alpine caves, Nitrosopumi- lus and Candidatus Nitrososphaera have been identified THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY ACTA CARSOLOGICA 49/2-3 – 2020 289 as dominant genus (Reitschuler et al. 2016). It is sug- gested that these microorganisms can provide nitrogen, through ammonium oxidation, to the other Dupnisa Cave organisms. Consequently, they contribute to the nutrient cycle of the cave. In parallel with the results of our study, AOA were found to be more dominant than ammonia oxidizing bacteria in Heshang Cave and Wee- bubbie Cave in China (Tetu et al. 2013; Zhao et al. 2017). It was suggested that the high level of AOA results from the high substrate affinity that allows them to multiply at lower ammonia concentrations than other organisms (Martens-Habbena et al. 2009). The other phylum belonging to the archaea found in the Dupnisa Cave is Euryarchaeota which is one of the dominant phyla of the cave ecosystem (Chelius & Moore 2004; Chen et al. 2009; Engel et al. 2010; Yasir 2018; Wiseschart et al. 2018). Methanogens in the Eu- ryarchaeota phylum are anaerobic microorganisms with a unique metabolism that produces methane as a meta- bolic by-product. Methanothermus, Methanobrevibacter, Methanosaeta, Methanotorris, Methanococcus, Methano- bacterium, Methanochaldococcus genus, which are mem- bers of Euryarchaeota phylum observed in Dupnisa Cave System, were determined at different sample points at different rates. Iţcuş et al. (2016) found Methanobacte- rium genus in the ice sample from Scarisoara Ice Cave (Romania). Methanogens are widely distributed in hot, acidic, salty areas such as deep sea sediments, freshwa- ter sediments, rice field soils, hot springs and gut systems (Sarmiento et al. 2011; Ciais et al. 2014). The identifica- tion of thermophilic Methanogens in the Dupnisa Cave part opened for tourism, may be due to the large number of bats in that cave. It is also interesting to note that thermophiles such as the genus Thermococcus (4.5 %), Thermoplasma (3.3 %) and Thermogymnomonas (3.2 %), which are also members of the Euryarchaeota phylum, are present in the mesophilic environment of the Dupnisa Cave at a rate of 3 % or more at a single point. As observed in the AF sample of our sample, the genus Thermogymnomo- nas has been detected also in the Hundalm Cave in Tyrol (Austria) (Reitschuler et al. 2014). Although the Thermo- plasmata has been reported in other cave environments, their ecological role in these environments still unclear. Their presence in caves has been widely identified in the sulfide rich environments (Macalady et al. 2006; Jones et al. 2012; Jones et al. 2014; Gulecal-Pektas & Temel 2017). However, Thermoplasmata-related 16S rRNA sequences have been recently observed in all moonmilk (Reitschul- er et al. 2014; Reitschuler et al. 2015) and cave sediments on a dry stream bed (Zhao et al. 2017). As a result of metagenomic analysis of the samples taken from different points, the variation in the genus level became clear even though the difference in phylum level was not significant. Each microenvironment reveals its own microbial profile. According to the Chao 1 rich- ness estimation index, the highest bacterial richness was found at the AF point (Cave entrance / cave sediment) and the most archaeal types were at the F2 point (Tour- istic area 2 / cave sediment). The AF point is affected by daylight as well as bats and tourists. According to the Shannon-Wiener diversity index, the sampling point with the most abundant archaeal and bacterial genus was determined as F2. Dupnisa Cave has a rich content in terms of bac- terial and archaea variety. The river running through it, guanos and tourists that visit there could be the source of the organic material inputs of Dupnisa Cave. When the bacteria and archaea taxa, which were determined in our study and are found predominantly in all caves, are examined at the genus level, it is seen that even the differ- ent points of each cave do not resemble each other. The diversity of bacteria and archaea revealed in the samples taken from the part open to tourism and the closed part of the Dupnisa Cave is a good example to prove it. Al- though the bacteria and archaea species detected in the Dupnisa Cave were found in other caves with different physicochemical properties, the communities formed by these genera and their percentages are different from each other and this is one of the most important factors that make the caves unique. CONCLUSIONS The caves are often difficult to be accessed but are ideal underground habitats for studying microbial me- tabolisms and microorganism relationships under oligo- trophic conditions. Number of studies in microbiological diversity carried out in our days is increasing day by day, while the studies in this field were previously limited. Al- though the number of studies is increasing, each cave is unique in parallel with its features, and the studies con- tinue to maintain their values. With this study, a molecu- lar independent bacterial and archaeal profile work was performed for the first time in Dupnisa Cave. According to the obtained results, difference between cave parts, in NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 290 term of microbial diversity, is revealed. Microbial biodi- versity studies are important for us, since it provides new and different data which contribute to the world of mi- croorganisms or this reason, the research of the Dupnisa Cave System is a guide for future studies. Open or closed to tourism, our results show that the bacterial and archaeal communities in Dupnisa Cave are composed of niche-specific members rather than cos- mopolitan. The high population of ammonia-oxidizing archaea (AOA) discovered in our study suggests based on microbial diversity analysis that the ammonium oxi- dation could be one of the main energy sources in the Dupnisa Cave ecosystem. Our future studies will be aimed to enrich the biodiversity information and to de- termine the microorganisms with enzyme / antibiotic potential by adding samples from the walls of Dupnisa Cave System. This study is not a complete picture of the microbial diversity of the Dupnisa Cave. We believe that these findings and our next studies will contribute to reach the big picture. ACKNOWLEDGMENTS The author would like to thank the Anatolian Spe- leology Association for sampling. The authors also thank Istanbul University Scientific Project Unit (BAP Project no 29244 and 42517) for their financial support. REFERENCES Anda, D., Krett, G., Makk, J., Márialigeti, K., Mádl- Szőnyi, J. & A.K. Borsodi, 2017: Comparison of Bacterial and Archaeal communities from different habitats of the hypogenic Molnár János Cave of the Buda Thermal Karst System (Hungary).- Journal of Cave and Karst Studies, 79, 2, 113-121. https://doi. org/10.4311/2015MB0134 Barton, H.A., Taylor, M.R. & N.R. Pace, 2004: Molecu- lar phylogenetic analysis of a bacterial commu- nity in an oligotrophic cave environment.- Ge- omicrobiology Journal, 21, 1, 11-20. https://doi. org/10.1080/01490450490253428 Barton, H.A., 2006: Introduction to cave microbiology: a review for the non-specialist.- Journal of Cave and Karst Studies, 68, 2, 43–54. Barton, H.A. & V. Jurado, 2007: What’s up down there? Microbial diversity in caves.- Microbe, 2, 132–138. Barton, H.A., Taylor, N.M., Kreate, M.P., Springer, A.C., Oehrle, S.A. & J.L. Bertog, 2007: The impact of host rock geochemistry on bacterial community struc- ture in oligotrophic cave environments.- Interna- tional Journal of Speleology, 36, 2, 93-104. http:// dx.doi.org/10.5038/1827-806X.36.2.5 Barton, H.A., 2015: Starving artists: Bacterial oligotroph- ic heterotrophy in caves.- In: Engel A. & De Gruyter W. (eds.) Life in Extreme Environments: Microbial Life of Cave Systems. De Gruyter, pp. 350, New Y ork. Bartossek, R., Spang, A., Weidler, G., Lanzen, A. & C. Schleper, 2012: Metagenomic analysis of ammonia- oxidizing archaea affiliated with the soil group.- Frontiers in microbiology, 3, 208. https://doi. org/10.3389/fmicb.2012.00208 Baskar, S., Chalia, S. & R. Baskar, 2018: Calcite precipi- tation by Rhodococcus sp. isolated from Kotumsar cave, Chhattisgarh, India.- Current Science, 114, 5, 1063-1074.   https://doi.org/10.18520/cs/v114/ i05/1063-1074 Bastian, F. & C. Alabouvette, 2009: Lights and shadows on the conservation of a rock art cave: the case of Lascaux Cave.- International Journal of Speleol- ogy, 38, 1, 55-60. http://dx.doi.org/10.5038/1827- 806X.38.1.6 Bates, S.T., Berg-Lyons, D., Caporaso J.G., Walters, W .A., Knight, R. & N. Fierer, 2011: Examining the global distribution of dominant archaeal populations in soil.- The ISME journal, 5, 908-917. https://doi. org/10.1038/ismej.2010.171 Carmichael, M.J., Carmichael, S.K., Santelli, C.M., Strom, A. & S.L. Bräuer, 2013: Mn (II)-oxidizing bacteria are abundant and environmentally relevant mem- bers of ferromanganese deposits in caves of the up- per Tennessee River Basin.- Geomicrobiology Jour- nal, 30, 9, 779-800. https://doi.org/10.1080/014904 51.2013.769651 Chelius, M.K. & J.C. Moore, 2004: Molecular phylogenet- ic analysis of Archaea and Bacteria in Wind Cave, South Dakota.- Geomicrobiology Journal, 21, 2, 123- 134. https://doi.org/10.1080/01490450490266389 THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY ACTA CARSOLOGICA 49/2-3 – 2020 291 Chelius, M.K., Beresford, G., Horton, H., Quirk, M., Selby, G., Simpson, R.T., Horrocks, R. & J.C. Moore, 2009: Impacts of alterations of organic inputs on the bacterial community within the sediments of Wind Cave, South Dakota, USA.- International Journal of Speleology, 38, 1-10. https://dx.doi. org/10.5038/1827-806X.38.1.1 Chen, Y., Wu, L., Boden, R., Hillebrand, A., Kumaresan, D., Moussard, H., Baciu, M., Lu, Y. & J.C. Murrell, 2009: Life without light: microbial diversity and evi- dence of sulfur- and ammonium-based chemolitho- trophy in Movile Cave.- The ISME Journal, 3, 1093- 1104. https://doi.org/10.1038/ismej.2009.57 Chroňáková, A., Horák, A., Elhottová, D. & V . Krištůfek, 2009: Diverse archaeal community of a bat guano pile in Domica Cave (Slovak Karst, Slovakia).- Fo- lia microbiologica, 54, 5, 436-446. https://doi. org/10.1007/s12223-009-0061-2  Ciais, P ., C. Sabine, G. Bala, L. Bopp, V . Brovkin, J. Can- adell, A. Chhabra, R. DeFries, J. Galloway, M. Hei- mann, C. Jones, C. Le Quéré, R.B. Myneni, S. Piao & P . Thornton, 2013: Carbon and Other Biogeochemi- cal Cycles Supplementary Material.- In: Stocker, T.F. et al. (eds.) Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmen- tal Panel on Climate Change. Cambridge University Press, pp. 465-570, Cambridge. Cuezva, S., Fernandez-Cortes, A., Porca, E., Pašić, L., Ju- rado, V., Hernandez-Marine, M., Serrano-Ortiz, P., Hermosin, B., Cañaveras, J.C., Sanchez-Moral, S. & C. Saiz-Jimenez, 2012: The biogeochemical role of actinobacteria in Altamira cave, Spain.- FEMS microbiology ecology, 81, 1, 281-290. https://doi. org/10.1111/j.1574-6941.2012.01391.x De Mandal, S., Panda, A.K., Bisht, S.S. & N.S. Kumar, 2015: First report of bacterial community from a bat guano using Illumina next-generation sequencing.- Genomics Data, 4, 99-101. https://doi.org/10.1016/j. gdata.2015.04.001 De Leon, M.P., Montecillo, A.D., Pinili, D.S., Siringan, M.A.T. & D.S. Park, 2018: Bacterial diversity of bat guano from Cabalyorisa Cave, Mabini, Pangasinan, Philippines: A first report on the metagenome of Philippine bat guano.- PloS one, 13, 10, e0200095. https://doi.org/10.1371/journal.pone.0200095 Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C. & R. Knight, 2011: UCHIME improves sensitivity and speed of chimera detection.- Bioinformatics, 27, 16, 2194-2200. https://doi.org/10.1093/bioinformatics/ btr381 Engel, A.S., 2010: Microbial diversity of cave ecosystem.- In: Loy A. et al. (eds.) Geomicrobiology: Molecular and environmental perspective. Springer Science and Business Media B.V ., pp. 219-238, Dordrecht. Engel, A.S., Meisinger, D.B., Porter, M.L., Payn, R.A., Schmid, M., Stern, L.A., Schleifer, K.H. & N.M. Lee, 2010: Linking phylogenetic and functional diversity to nutrient spiraling in microbial mats from Lower Kane Cave (USA).- The ISME journal, 4, 98-110. https://doi.org/10.1038/ismej.2009.91 Fong, D.W ., 2011: Management of subterranean fauna in karst.- In: Van Beynen P .E. (ed.) Karst management. Springer, pp. 201-224, Dordrecht.. General Directorate of Mineral Research And Explora- tions, 2020: Karst cave in Turkey and inventory.- [Online] Available from: https://www.mta.gov.tr/ v3.0/arastirmalar/magara-envanteri [Accessed 3rd April 2020]. Griffin, D.W., Gray, M.A., Lyles, M.B. & D.E. Northup, 2014: The transport of nonindigenous microorgan- isms into caves by human visitation: a case study at Carlsbad Caverns National Park.- Geomicrobiology Journal, 31, 3, 175-185. https://doi.org/10.1080/014 90451.2013.815294 Groth, I. & C. Saiz-Jiménez, 1999: Actinomycetes in hypo- gean environments.- Geomicrobiology Journal, 16, 1, 1-8. https://doi.org/10.1080/014904599270703 Groth, I., V ettermann, R., Schuetze, B., Schumann, P . & C. Saiz-Jiménez, 1999: Actinomycetes in karstic caves of Northern Spain (Altamira and Tito Bustillo).- Journal of Microbiological Methods, 36, 1-2, 115- 122. https://doi.org/10.1016/S0167-7012(99)00016- 0 Gulecal-Pektas, Y., 2016: Bacterial diversity and com- position in oylat cave (Turkey) with combined sanger/pyrosequencing approach.- Polish Jour- nal of Microbiology, 65, 1, 69-75. https:77doi. org/10.5604/17331331.1197277 Gulecal-Pektas, Y. & M. Temel, 2017: A Window to the Subsurface: Microbial Diversity in Hot Springs of a Sulfidic Cave (Kaklik, Turkey).- Geomicrobiology Journal, 34, 4, 374-384. https://doi.org/10.1080/014 90451.2016.1204374 Hathaway, J.J.M., Garcia, M.G., Balasch, M.M., Spilde, M.N., Stone, F.D., Dapkevicius, M.D.L.N.E., Amor- im, I.R., Gabriel, Borges, P.A.V. & D.E. Northup, 2014: Comparison of bacterial diversity in Azorean and Hawai'ian lava cave microbial mats.- Geomi- crobiology journal, 31, 3, 205-220. https://doi.org/ 10.1080/01490451.2013.777491 Hoyos, M., Soler, V., Cañaveras, J.C., Sánchez-Moral, S. & E. Sanz-Rubio, 1998: Microclimatic characteriza- tion of a karstic cave: human impact on microenvi- ronmental parameters of a prehistoric rock art cave (Candamo Cave, northern Spain).- Environmental NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 292 Geology, 33, 4, 231-242. https://doi.org/10.1007/ s002540050242 Ikner, L.A., Toomey, R.S., Nolan, G., Neilson, J.W., Pry- or, B.M. & R.M. Maier, 2007: Culturable microbial diversity and the impact of tourism in Kartchner Caverns, Arizona.- Microbial Ecology, 53, 30-42. https://doi.org/10.1007/s00248-006-9135-8 Iţcuş, C., Pascu, M.D., Brad, T., Perşoiu, A., & C. Purcar- ea, 2016: Diversity of cultured bacteria from the pe- rennial ice block of Scarisoara Ice Cave, Romania.- International Journal of Speleology, 45, 1, 89-100. http://dx.doi.org/10.5038/1827-806X.45.1.1948 Jones, D.S., Tobler, D.J., Schaperdoth, I., Mainiero, M. & J.L. Macalady, 2010: Community structure of subsurface biofilms in the thermal sulfidic caves of Acquasanta Terme, Italy.- Applied and Environ- mental Microbiology, 76, 17, 5902-5910. https://doi. org/10.1128/AEM.00647-10 Jones, D.S., Albrecht, H.L., Dawson, K.S., Schaperdoth, I., Freeman, K.H., Pi Y., Pearson, A. & J.L. Maca- lady, 2012: Community genomic analysis of an ex- tremely acidophilic sulfur-oxidizing biofilm.- The ISME Journal, 6, 158-170. https://doi.org/10.1038/ ismej.2011.75 Jones, A.A. & P.C. Bennett, 2014: Mineral microniches control the diversity of subsurface microbial popu- lations.- Geomicrobiology Journal, 31, 3, 246-261. https://doi.org/10.1080/01490451.2013.809174 Jurado, V ., Laiz, L., Rodriguez-Nava, V ., Boiron, P ., Her- mosin, H., Sanchez-Moral, S. & C., Saiz-Jimenez, 2010: Pathogenic and opportunistic microorgan- isms in caves.- International Journal of Speleol- ogy, 39, 1, 15-24. http://dx.doi.org/10.5038/1827- 806X.39.1.2 Kalyuzhnaya, M.G., Lidstrom, M.E. & L. Chistoserdova, 2008: Real-time detection of actively metabolizing microbes by redox sensing as applied to methylo- troph populations in Lake Washington.- The ISME Journal, 2, 696-706. https://doi.org/10.1038/is- mej.2008.32 Kersters, K., De Vos, P ., Gillis, M., Swings, J., Vandamme, P. & E. Stackebrandt, 2006: Introduction to the Proteobacteria.- In: Dworkin, M. et al. (eds.) The Prokaryotes: a handbook on the biology of bacteria, 3rd Ed. Springer‐Verlag, pp. 3-37, New Y ork. Kieraite-Aleksandrova, I., Aleksandrovas, V. & N. Kuisiene, 2015: Down into the Earth: microbial diversity of the deepest cave of the world.- Bio- logia, 70, 8, 989-1002. https://doi.org/10.1515/bi- olog-2015-0127 Kimble, J.C., 2017: Comparison of Bacterial and Archaeal Communities in the Subsurface versus Surface: Impli- cations for Nitrogen Cycling.- PhD thesis. University of New Mexico, pp.152. Lavoie, K.H., Winter, A.S., Read, K.J., Hughes, E.M., Spilde, M.N. & D.E. Northup, 2017: Comparison of bacterial communities from lava cave microbial mats to overlying surface soils from Lava Beds Na- tional Monument, USA.- PloS one, 12, 2, e0169339. https://doi.org/10.1371/journal.pone.0169339 Lee, I.T., Liu, J.Y., Lin, C.H., Oyama, K.-I., Chen, C.Y. & C.H. Chen, 2012: Ionospheric plasma caves under the equatorial ionization anomaly.- Journal of Geo- physical Research, 117, A11, A11309, https://doi. org/10.1029/2012JA017868 Macalady, J.L., Lyon, E.H., Koffman, B., Albertson, L.K., Meyer, K., Galdenzi, S. & S. Mariani, 2006: Domi- nant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy.- Applied and Environmental Microbiology, 72, 8, 5596-5609. https://doi.org/10.1128/AEM.00715-06 Martens-Habbena, W., Berube, P.M., Urakawa, H., José, R. & D.A. Stahl, 2009: Ammonia oxidation kinet- ics determine niche separation of nitrifying Archaea and Bacteria.- Nature, 461, 976-979. https://doi. org/10.1038/nature08465 Mulec, J. & G. Kosi, 2009: Lampenflora algae and meth- ods of growth control.- Journal of cave and karst studies, 71, 2, 109-115. Nazik, L., Törk K., Özel, E., Mengi, H., Aksoy, B. & C. Acar, 1998: Kuzey ve Kuzeydoğu Trakya’nın (Kırklareli-Tekirdağ) Doğal Mağaraları. Dosya No: 43584. Maden Tetkik ve Arama Genel Müdürlüğü, Jeoloji Etütleri Dairesi Başkanlığı, Ankara. Northup D.E. & K.H. Lavoie, 2001: Geomicrobiology of caves: a review .- Geomicrobiology Journal, 18, 3, 199- 222. https://doi.org/10.1080/01490450152467750 Northup, D.E., Barns, S.M., Yu, L.E., Spilde, M.N., Schel- ble, R.T., Dano, K.E., Crossey, L.J., Connolly, C.A., Boston, P.J., Natvig, D.O. & C.N. Dahm, 2003: Di- verse microbial communities inhabiting ferroman- ganese deposits in Lechuguilla and Spider Caves.- Environmental Microbiology, 5, 11, 1071-1086. https://doi.org/10.1046/j.1462-2920.2003.00500.x Oliveira, C., Gunderman, L., Coles, C.A., Lochmann, J., Parks, M., Ballard, E., Glazko, G., Rahmatallah, Y., Tackett, A.J. & D.J. Thomas, 2017: 16S rRNA Gene- Based Metagenomic Analysis of Ozark Cave Bac- teria.- Diversity, 9, 3, 31. https://doi.org/10.3390/ d9030031 Ortiz, M., Neilson, J.W ., Nelson, W .M., Legatzki, A., By- rne, A., Yu, Y., Wing, R.A., Soderlund, C.A., Pryor, B.M., Pierson, L.S. & R.M. Maier, 2013: Profil- ing bacterial diversity and taxonomic composi- tion on speleothem surfaces in Kartchner Caverns, THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY ACTA CARSOLOGICA 49/2-3 – 2020 293 AZ.- Microbial ecology, 65, 371-383. https://doi. org/10.1007/s00248-012-0143-6 Ortiz, M., Legatzki, A., Neilson, J.W., Fryslie, B., Nel- son, W.M., Wing, R.A., Soderlund, C., Pryor, B.M. & R.M. Maier, 2014: Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave.- The ISME Journal, 8, 478-491. https://doi.org/10.1038/ismej.2013.159 Palmer A.N., 1991: Origin and morphology of lime- stone caves.- Geological Society of America Bul- letin, 103, 1, 1–21. https://doi.org/10.1130/0016- 7606(1991)103<0001:OAMOLC>2.3.CO;2 Paksuz, S., Özkan, B., & T. Postawa, 2007: Seasonal changes of cave-dwelling bat fauna, and their rela- tionship with microclimate in Dupnisa Cave System (Turkish Thrace).- Acta Zoologica Cracoviensia- Series A: Vertebrata, 50, 1-2, 57-66. https://doi. org/10.3409/000000007783995435 Paksuz, S., 2017: Important Caves in Turkish Thrace for Bats: Dupnisa Cave System and Koyunbaba Cave.- In: S. Karabulut & M. Cengiz (eds) Cave Investiga- tion. IntechOpen, pp. 77-93, London. Pašić, L., Kovče, B., Sket, B., & B. Herzog-V elikonja, 2010: Diversity of microbial communities colonizing the walls of a Karstic cave in Slovenia.- FEMS Microbi- ology Ecology, 71, 1, 50-60. https://doi.org/10.1111/ j.1574-6941.2009.00789.x Pester, M., Schleper, C., & M. Wagner, 2011: The Thaumarchaeota: an emerging view of their phy- logeny and ecophysiology.- Current opinion in mi- crobiology, 14, 3, 300-306. https://doi.org/10.1016/j. mib.2011.04.007 Porter, M.L., Engel, A.S., Kane, T.C. & B.K. Kinkle, 2009: Productivity-diversity relationships from chemo- lithoautotrophically based sulfidic karst systems.- International Journal of Speleology, 38, 1, 27-40. http://dx.doi.org/10.5038/1827-806X.38.1.4 Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W ., Peplies, J. & F.O. Glöckner, 2007: SILV A: a com- prehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB.- Nucleic Acids Research, 35, 21, 7188- 7196. https://doi.org/10.1093/nar/gkm864 Reitschuler, C., Lins, P ., Wagner, A. O. & P . Illmer, 2014: Cultivation of moonmilk-born non-extremophilic Thaum and Euryarchaeota in mixed culture.- An- aerobe, 29, 73-79. https://doi.org/10.1016/j.anaer- obe.2013.10.002 Reitschuler, C., Lins, P., Schwarzenauer, T., Spötl, C., Wagner, A.O. & P. Illmer, 2015: New undescribed lineages of non-extremophilic archaea form a ho- mogeneous and dominant element within alpine moonmilk microbiomes.- Geomicrobiology Jour- nal, 32, 10, 90-902. https://doi.org/10.1080/014904 51.2015.1025317 Reitschuler, C., Spötl, C., Hofmann, K., Wagner, A.O. & P. Illmer, 2016: Archaeal distribution in moonmilk deposits from Alpine caves and their ecophysi- ological potential.- Microbial ecology, 71, 686-699. https://doi.org/10.1007/s00248-015-0727-z Rusznyák, A., Akob, D.M., Nietzsche, S., Eusterhues, K., Totsche, K.U., Neu, T.R., Frosch, T., Popp, J., Keiner, R., Geletneky, J., Katzschmann, L., Schulze, E.D. & L. Küsel, 2012: Calcite biomineralization by bacte- rial isolates from the recently discovered pristine karstic Herrenberg cave.- Applied and Environ- mental Microbiology, 78, 1157-1167. https://doi. org/10.1128/AEM.06568-11 Sarmiento, F.B., Leigh, J.A., & W .B. Whitman, 2011: Ge- netic systems for hydrogenotrophic methanogens.- In: Rosenzweig, A.C. et al. (eds.) Methods in enzy- mology. Academic Press, 494, 43-73. Schabereiter-Gurtner, C., Saiz-Jimenez, C., Piñar, G., Lu- bitz, W . & S. Rölleke, 2002: Altamira cave Paleolithic paintings harbor partly unknown bacterial commu- nities.- FEMS Microbiology Letters, 211, 1, 7-11. https://doi.org/10.1111/j.1574-6968.2002.tb11195.x Shapiro J. & A. Pringle, 2010: Anthropogenic influences on the diversity of fungi isolated from caves in Ken- tucky and Tennessee.- The American Midland Nat- uralist, 163, 1, 76-87. https://doi.org/10.1674/0003- 0031-163.1.76 Stahl, D.A. & J.R. De la Torre, 2012: Physiology and di- versity of ammonia-oxidizing archaea.- Annual review of microbiology, 66, 83-101. https://doi. org/10.1146/annurev-micro-092611-150128 Tetu, S.G., Breakwell, K., Elbourne, L.D., Holmes, A.J., Gillings, M.R. & I.T. Paulsen, 2013: Life in the dark: metagenomic evidence that a microbial slime com- munity is driven by inorganic nitrogen metabo- lism.- The ISME journal, 7, 1227-1236. https://doi. org/10.1038/ismej.2013.14 The Human Microbiome Project Consortium, 2012: Structure, function and diversity of the healthy hu- man microbiome.- Nature, 486, 207-214. https:// doi.org/10.1038/nature11234 Tomczyk-Żak, K. & U. Zielenkiewicz, 2016: Microbial diversity in caves.- Geomicrobiology Journal, 33, 1, 20-38. https://doi.org/10.1080/01490451.2014.1003 341 Torsvik, V. & L. Øvreås, 2002: Microbial diversity and function in soil: from genes to ecosystems.- Current Opinion in Microbiology, 5, 3, 240-245. https://doi. org/10.1016/S1369-5274(02)00324-7 Van Dijk, E.L., Auger, H., Jaszczyszyn, Y. & C. Thermes, 2014: T en years of next-generation sequencing tech- NIHAL DOĞRUÖZ-GÜNGÖR ACTA CARSOLOGICA 49/2-3 – 2020 294 nology.- Trends in genetics, 30, 9, 418-426. https:// doi.org/10.1016/j.tig.2014.07.001 Wiseschart, A., Mhuanthong, W., Thongkam, P., Tang- phatsornruang, S., Chantasingh, D. & K. Pootanakit, 2018: Bacterial Diversity and Phylogenetic Analysis of Type II Polyketide Synthase Gene from Manao- Pee Cave, Thailand.- Geomicrobiology Journal, 35, 6, 518-527. https://doi.org/10.1080/01490451.2017. 1411993 Wu, J.H., Chen, W .Y., Kuo, H.C. & Y.M. Li, 2019: Redox fluctuations shape the soil microbiome in the hy- poxic bioremediation of octachlorinated dibenzo- dioxin-and dibenzofuran-contaminated soil.- En- vironmental Pollution, 248, 506-515. https://doi. org/10.1016/j.envpol.2019.02.053 Y an, Y ., Fu, D. & J. Shi, 2019: Screening and Immobilizing the Denitrifying Microbes in Sediment for Bioreme- diation.- Water, 11, 3, 614. https://doi.org/10.3390/ w11030614 Yasir, M., 2018: Analysis of bacterial communities and characterization of antimicrobial strains from cave microbiota.- Brazilian Journal of Microbi- ology, 49, 2, 248-257. https://doi.org/10.1016/j. bjm.2017.08.005 Zhou, J., Gu, Y., Zou, C. & M. Mo, 2007: Phylogenetic diversity of bacteria in an earth-cave in Guizhou Province, Southwest of China.- Journal of Microbi- ology, 45, 2, 105-112. Zhao, R., Wang, H., Yang, H., Yun, Y. & H.A. Barton, 2017: Ammonia-oxidizing Archaea dominate am- monia-oxidizing communities within alkaline cave sediments.- Geomicrobiology journal, 34, 6, 511- 523. https://doi.org/10.1080/01490451.2016.12258 61 THE MICROBIAL COMMUNITY STRUCTURE OF THE DUPNISA CAVE IN KIRKLARELI, TURKEY ACTA CARSOLOGICA 49/2-3 – 2020 295