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Biofilm Bacterial Communities Inhabiting the CaveWalls of the Buda Thermal Karst System, HungaryAndrea K. Borsodi a , Mónika Knáb a , Gergely Krett a , Judit Makk a , Károly Márialigeti a ,Anita Erőss b & Judit Mádl-Szőnyi b

a Department of Microbiology, Eötvös Loránd University, Budapest, Hungaryb Department of Physical and Applied Geology, Eötvös Loránd University, Budapest, Hungary

Available online: 05 Jun 2012

To cite this article: Andrea K. Borsodi, Mónika Knáb, Gergely Krett, Judit Makk, Károly Márialigeti, Anita Erőss & JuditMádl-Szőnyi (2012): Biofilm Bacterial Communities Inhabiting the Cave Walls of the Buda Thermal Karst System, Hungary,Geomicrobiology Journal, 29:7, 611-627

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Geomicrobiology Journal, 29:611–627, 2012Copyright © Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451.2011.602801

Biofilm Bacterial Communities Inhabiting the Cave Wallsof the Buda Thermal Karst System, Hungary

Andrea K. Borsodi,1 Monika Knab,1 Gergely Krett,1 Judit Makk,1

Karoly Marialigeti,1 Anita Eross,2 and Judit Madl-Szonyi2

1Department of Microbiology, Eotvos Lorand University, Budapest, Hungary2Department of Physical and Applied Geology, Eotvos Lorand University, Budapest, Hungary

The diversity of biofilm bacterial communities associated withcave walls of the Buda Thermal Karst System (BTKS) located inHungary was studied by scanning electron microscopy and molec-ular cloning based on 16S rRNA genes. Samples from two sites,the Molnar Janos cave (MJB) and the Rudas-Torok spring cave(RTB), respectively, were analyzed and compared. The presence ofiron precipitates was typical at both study sites, despite the factthat the cell morphological structure of the biofilms observed bySEM was characteristically different. Clones analyzed from BTKSwere found to belong to 10 common phyla (Thermodesulfobac-teria, Chloroflexi, Nitrospirae, Chlorobi, Proteobacteria, Firmi-cutes, Actinobacteria, Planctomycetes, Bacteroidetes, Verrucomi-crobia) within the domain Bacteria. Moreover, sequences related toAquificeae, Acidobacteria and Gemmatimonadetes were exclusiveto MJB, while Cyanobacteria were found in RTB only. The phy-logenetic distribution of the dominant bacterial clones was quitedissimilar between the two sites. In the biofilm from MJB clonesaffiliated with Firmicutes, whereas in the RTB clones related toDeltaproteobacteria were found in the highest number. In addi-tion, substantially larger numbers of clone sequences related tothermophilic bacteria were recovered from MJB. On the basis ofsequences of known microorganisms corresponding to our clonesequences, it is assumed that aerobic as well as anaerobic iron andsulfur transformation performed by different bacterial communi-ties might be important biogenic processes in both caves.

Keywords karst cave, biofilm, bacterial diversity, SEM, clone library,16S rDNA

Received 5 April 2011; accepted 28 June 2011.This study was accomplished within the framework of the col-

laboration between Shell International E&P and the Eotvos LorandUniversity. The European Union and the European Social Fund havealso provided financial support to the project under the grant agree-ment no. TAMOP 4.2.1./B-09/KMR-2010-0003. This research wasalso supported by the Hungarian Scientific Research Fund (OTKA)Grant NK101356.

Address correspondence to Andrea K. Borsodi, Eotvos Lorand Uni-versity, Department of Microbiology, Pazmany Peter setany 1/C, 1117Budapest, Hungary. E-mail: bandrea@caesar.elte.hu

INTRODUCTIONOver the last decades, several studies have been published

in order to report on the microorganisms living in or contam-inated caves and/or with the purpose of exploring the possiblerole of microorganisms in recent cave formations (Barton andNorthup 2007). Due to the strong selectivity and inadequacy ofthe cultivation based techniques, mainly molecular biologicaltechniques helped reveal an enormous extent of microbial di-versity associated worldwide with different caves (Barton et al.2007; Canaveras et al. 2001; Chelius and Moore 2004; Engelet al. 2001; Groth et al. 2001; Holmes et al. 2001; Macaladyet al. 2006, 2007; Northup et al. 2000, 2003; Pasic et al. 2010;Porter et al. 2009; Portillo et al. 2009; Schabereiter-Gurtner et al.2002; Zhou et al. 2007).

Regardless of the media used for cultivation, among theaerobic heterotrophic bacteria recovered, Actinobacteria werefound to be the most abundant in caves from rock walls andbiofilms (Canaveras et al. 2001; Chelius and Moore 2004; Grothet al. 2001). Further cultivated strains were members of differentAlpha-, Beta- and Gammaproteobacteria, including Thiobacil-lus related sulfur-oxidizing bacteria (Chelius and Moore 2004;Engel et al. 2001). In almost all clone libraries of differentcave environments (e.g., Earth-cave, Guizhou Province, China;Frasassi cave system, Italy; Lechuguilla and Spider Caves,USA; Nullarbor caves, Australia; Pajsarjeva jama, Slovenia;Tito Bustillo cave, Spain; Wind Cave, USA), members of Pro-teobacteria (in most cases Gammaproteobacteria) formed thepredominant group (Holmes et al. 2001; Macalady et al. 2006,2007; Northup et al. 2003; Pasic et al. 2010; Schabereiter-Gurtner et al. 2002; Zhou et al. 2007). In microbial mats fromsome active sulfidic caves located in Romania, Italy and USAchemolithotrophic bacteria affiliated with Epsilonproteobacteria(Porter et al. 2009) were the highest number of clones recovered.Additional prevalent clones detected from cave environmentswere affiliated with Acidobacteria (Chelius and Moore 2004;Pasic et al. 2010; Schabereiter-Gurtner et al. 2002; Zhou et al.2007), Nitrospirae (Holmes et al. 2001; Pasic et al. 2010) andActinobacteria (Northup et al. 2003; Pasic et al. 2010).

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612 A. K. BORSODI ET AL.

Due to carbonate dissolution, the participation of mainlychemolithoautotrophic microorganisms (e.g. members ofEpsilon- and Gammaproteobacteria), was presumed in sulfuricacid based hypogenic speleogenesis. In recent hypogenic karststudies, not only microorganism-mediated oxidation and reduc-tion of sulfuric compounds, but methanogenesis, as well as metal(Fe and Mn) oxidation and reduction have been demonstrated,which can all facilitate carbonate dissolution by influencingthe local proton concentration relationships (Bennett and Engel2005; Engel and Randall 2008).

Furthermore, the accumulation of radium, causing high ra-dioactivity, was observed in iron and manganese-containing mi-crobial mats of hot springs in Japan. Beside radium, the accumu-lation of arsenic and other trace elements through precipitationand complexation on the bacterial cell surface was also reported(Fujisawa and Tazaki 2003). Microbially controlled recent fer-rihydrite and carbonate precipitation was also described fromthe Cezallier spring (Massif Central, France) by Casanova et al.(1999) and Le Guern et al. (2003).

Budapest is the capital of spas and Europe’s largest natu-rally flowing thermal water system (the hydrogeologically ac-tive Buda Thermal Karst System, BTKS), which provides manypossibilities for studying microorganisms inhabiting cave envi-ronments. The BTKS belongs to the large carbonate rock aquifersystem of the Transdanubian Central Range in Hungary (Fig. 1).One of the main discharge zones of its regional fluid flow can befound in Budapest, where springs with various temperatures andchemical composition discharge along the Danube. The mixingof ascending thermal waters, rich in dissolved solids, and de-scending meteoric waters resulted in the formation of spaciouscave systems.

The recharge area of the northern discharge zone, the so-called Rose Hill, is composed of large exposed carbonate sur-

faces facilitating the recharge of large amounts of meteoric fluid.Due to the confined conditions of Rose Hill, the discharge ofthe meteoric and hydrothermal fluids is structurally controlledand manifested in the forms of spatially separated lukewarm(20–35◦C) and hot (40–65◦C) springs (Eross et al. 2008a).

Due to the fact that the southern system, the Gellert Hill area,can be characterized with a limited surface of exposed carbon-ates, the meteoric fluid contribution is limited here; therefore thedischarged fluids mainly consist of upwelling hydrothermal wa-ters and lukewarm springs cannot be found here. Regarding themain components, the waters of the southern system are charac-terized by elevated Ca2+, Mg2+, HCO3

− and SO42−; therefore,

the total dissolved solid content is elevated in comparison withthe northern system (Fig. 2). These higher values within a nar-rower range (30–47◦C) correspond to lower temperatures thanthe hot waters of the northern system. However, there is no dif-ference between the two systems with regard to Na+ and Cl−

(Eross et al. 2008a, 2008b).In order to explore the putative bacterial activity related to

the dissolution and/or the precipitation of carbonate as well asiron and sulfur compounds, the present study had two objectives(a) to characterize cell morphological structures and analyze thechemical element composition of biofilms associated with theiron hydroxide precipitates found on the walls of two caveslocated in the Buda Thermal Karst System and (b) to reveal andcompare the bacterial diversity of biofilms by using 16S rRNAgene-based molecular phylogenetic techniques.

MATERIALS AND METHODS

Study Sites and SamplingsTwo representative sampling sites were chosen (Fig. 1) in the

area of the Buda Thermal Karst System (BTKS); the northern

FIG. 1. Location of the Buda Thermal Karst System in the Transdanubian Central Range and the sampling sites in Budapest (1, Subsurface boundary of Mesozoiccarbonates; 2, Uncovered Mesozoic carbonates; 3, Buda Thermal Karst System; a, Northern system, Rose Hill; b, Southern system, Gellert Hill).

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BIOFILM BACTERIAL COMMUNITIES 613

FIG. 2. Stiff diagram illustrating the distribution of main components of the cave-filling waters found at the MJB and RTB sampling sites.

Molnar Janos cave (MJB) at Rose Hill (N: 47.518; E: 19.036),and the southern Rudas-Torok spring cave (RTB) at Gellert Hill(N: 47.489; E: 19.046). The studied sites can not be visitedby tourists; therefore the direct human impact on the microbialdiversity is negligible. The host rocks in case of the MJB aremarl—sometimes with considerable carbonate content (up to80%)—and limestone while in case of the RTB is dolomite(Hauptdolomite). The main components of the cave waters aredemonstrated on a Stiff diagram (Fig. 2). The RTB samplingsite is characterized by higher dissolved solid content (TDS1400 mg l−1) than the MJB (TDS 873 mg l−1).

In addition to calcite, iron hydroxides (poorly crystallizedferrihydrite and goethite) are the most characteristic recent pre-cipitates of the discharge zone of BTKS. The iron is most proba-bly transported with the thermal water along regional flow paths(Eross et al. 2008a). There is a clear difference between theoccurrences of the iron hydroxides at the two study sites. In theMolnar Janos cave (MJB) at Rose Hill, these precipitates arefound in deep phreatic conditions on the cave walls, where theymay form by the mixing events of the reduced hydrothermaland the oxidative meteoric waters. In the Rudas-Torok springcave (RTB) at the Gellert Hill area iron hydroxides are lo-cated directly in the spring outlets under the water table, wherethe discharging reduced thermal waters may become oxidized(Eross et al. 2008a). These iron hydroxide precipitates are con-centrated in two-to-three-mm thick reddish-brown biofilms. Anapproximately 0.1 m2 biofilm-covered surface was sampled us-ing sterile scalpels at both sites. Samples were collected intosterile flasks and stored at 6–8◦C until being processed in thelaboratory within 4 h. At the time of sampling, the measuredwater temperature, pH and electric conductivity values were26.9◦C, 7.01 and 1068 µS cm−1 in the MJB, whereas 30.1◦C,6.93 and 1730 µS cm−1 in the RTB. Samples from both siteswere taken in March 2009 for scanning electron microscopy(SEM) and clone library construction.

Electron MicroscopyScanning electron microscopy was used to examine the mor-

phological structure of the biofilms in high resolution. First,500 µl of the samples was fixed in glutaraldehyde (5% in 0.1 Mphosphate buffer) for 3–4 hours. Fixed samples were then frozenin liquid nitrogen, freeze-dried, mounted and coated with gold.The samples were examined using a HITACHI S-2600N scan-ning electron microscope at an accelerating voltage of 20 kV.The microanalyses of the chemical element composition of thebiofilm samples were accomplished by EDS using an AM-RAY 1830 SEM with 20 kV accelerating voltage. The appliedAMRAY-1830 SEM is able to detect chemical elements of theperiodic table only after Na, hence the most important biogenicelements (C, N, O, H) are not appeared on the spectra.

Bacterial DNA Extraction and PCR AmplificationTotal community DNA extraction was carried out using the

Ultra Clean Soil DNA extraction kit (MO Bio Inc., CA, USA)according to the manufacturer’s instructions. Prior to DNA isola-tion biofilm samples was compacted by centrifugation (5000 rcffor 10 min). Isolated DNA was purified with GeneClean SpinKit (BIO 101 Inc., CA, USA) as specified by the manufacturerand detected in agarose gel (1%) stained with ethidium bromide,visualized with UV excitation.

16S rDNA was amplified by PCR using bacterial 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) primers (Lane 1991). The fol-lowing thermal profile was used in a Biometra T Personal ther-mal cycler (Biocompare, CA, USA): initial denaturation at 98◦Cfor 5 min, followed by 32 cycles consisting of denaturation at94◦C for 30 s, annealing at 52◦C for 30 s, and elongation at 72◦Cfor 1 min, followed by a final extension at 72◦C for 30 min.The PCR reaction mixture contained 200 mM of each deoxynucleoside triphosphate, 1 U of LC Taq DNA Polymerase(recombinant) (Fermentas, Lithuania), 1X Taq buffer with

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614 A. K. BORSODI ET AL.

(NH4)2SO4 (Fermentas, Lithuania), 2 mM MgCl2, 0.65 mM ofeach primer, and about 20 ng of genomic DNA template in a to-tal volume of 50 µL. 16S rDNA PCR products were purified andconcentrated using the PCR-M Clean Up Kit (Viogene-BiotekCorp., CA, USA) following the manufacturer’s instructions.

Construction of 16S rDNA Clone LibrariesThe PCR product was ligated into pGEM-T Vector Sys-

tem (Promega, WI, USA) and transformed into competentE. coli JM109 cells. The transformed cells were spread on Luria-Bertani plates containing 100 µg ml−1 ampicillin, 80 µg ml−1 X-Gal and 0.5 mM IPTG and incubated overnight at 37◦ C. Recom-binant plasmids were extracted from the E. coli cells by incu-bating the cultures at 98◦C for 5 min, and pelleting the cell frag-ments by centrifugation with 4500 rcf for 5 min. The inserts fromthe recombinant plasmids were amplified by PCR using standardplasmid-specific M13f (5′-GTAAAACGACGGCCAG-3′) andM13r (5′-CAGGAAACAGCTATGAC-3′) primer set (Messing1983). Reactions were incubated in a Biometra T Personal ther-mal cycler (Biocompare, CA, USA) as follows: denaturation at96◦C for 3 min, followed by 32 cycles consisting of denaturationat 94◦C for 30 sec, annealing at 52◦C for 30 sec and elongationat 72◦C for 1 min, and a final extension at 72◦C for 10 min. Toobtain the original inserts without the vector’s flanking regions,a nested PCR was carried out with the original primers (27f,1492r), and the following thermal protocol: initial denaturationat 96◦C for 3 min, followed by 32 cycles consisting of denatura-tion at 95◦C for 30 sec, annealing at 52◦C for 30 sec, elongationat 72◦C for 1 min, and a final extension at 72◦C for 10 minutes.

In order to group the clone sequences, Amplified RibosomalDNA Restriction Analysis (ARDRA) was carried out using theenzymes Hin6I and BsuRI (Fermentas, Lithuania) as describedby Massol-Deya et al. (1995). Digestion products were separatedin 2% ethidium-bromide-stained agarose gel (Gibco, CA, USA),visualized by UV excitation using a Micromax CCD camera, andanalyzed by image analyzer software (Princetone Instruments,NJ, USA).

Representatives of each ARDRA group were chosen fornucleotide sequence determination using the 534r primer (5′-ATTACCGCGGCTGCTGG-3′) (Muyzer et al. 1993) and theBig Dye Terminator Ready Cycle Sequencing Kit (AppliedBiosystems, CA, USA) as recommended by the manufacturer.Following ethanol-precipitation, sequences of approximately500 bp in the hypervariable (V3) region of the 16S rRNA genewere obtained with ABI PRISM 310 Genetic Analyzer (AppliedBiosystems, CA, USA).

All other sequences were screened for chimeras by the Mal-lard software (Ashelford et al. 2006), and chimeric sequencesand ambiguous sequences were excluded from further analysis.The uncultured bacterial partial 16S rRNA gene sequences weredeposited in GenBank under accession numbers FR754452-FR754485 for the MJB clones and FR754407-FR754451 forthe RTB clones.

Phylogenetic AnalysisThe diversity of clone libraries was investigated by rarefac-

tion analysis. Rarefaction curves were produced on a personalcomputer with the freeware Rarefaction Calculator program(http://www.biology.ualberta.ca/jbrzusto/rarefact.php#Credits).The expected number of different ARDRA patterns was plottedagainst the number of 16S rDNA clones. The number of dif-ferent ARDRA patterns in both clone libraries was determinedfollowing digestion with two restriction endonucleases.

Analysis of clone sequences and similarity search were per-formed with the BLAST algorithm (Basic Local Alignment andSearch Tool) on the NCBI (National Center for Biotechnol-ogy Information) nucleotide database (Altschul et al. 1997).The NCBI nucleotide database is known to contain a largenumber of sequences of uncultured bacteria and environmentalclones. Consequently, it is hard to gain data on the presumptivemetabolic potential and physiological properties of our clonesbased on their closest NCBI-BLAST hits. Therefore, all se-quences were aligned against the EzTaxon database (Chun et al.2007) of described and validated species and these closest cul-tivated species were also involved in the phylogenetic analysis.

RESULTS AND DISCUSSION

Electron Microscopic ObservationsOn the basis of images taken with SEM, there were con-

siderable morphological differences between the two biofilmsamples (Figs. 3A and 3B). In the MJB sample, the presenceof very similar straight or curved rod-shaped cells (length 1–3 µm, width 0.5–1 µm) and long filaments (length 15–20 µm,width 1 µm) were characteristic in homogeneous distributionbetween clay mineral crystals and small amounts of organicmatter matrix (Fig. 3A).

In the RTB sample, microbial cells with various morpholo-gies could be observed between small amounts of calcium-carbonate mineral crystals. Microbial cells were encompassedby a large amount of organic matter matrix which can be origi-nated from dead cells as well as extracellular polymer substances(Fig. 3B). Filamentous bacteria of different sizes (length 15–20 µm, width 0.5 and >1 µm) and morphologies often formedbundles; furthermore aggregates of straight or curved rodshaped-cells (length 1–3 µm, width 0.5–1 µm) were also char-acteristic.

Biofilm samples from MJB and RTB sites were also com-pared by SEM-EDX. Although the morphological structure ofthe samples from MJB and RTB were different, the EDX anal-ysis of both biofilm samples revealed the dominant presence ofiron oxides (Figs. 3C and 3D). The iron containing phases wereanalyzed with Mossbauer spectroscopy and found as ferrihydriteand poorly crystallized goethite.

Nevertheless, the appearance of hydrous iron oxides wasdissimilar inasmuch as very small crystalline forms covered the

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BIOFILM BACTERIAL COMMUNITIES 615

FIG. 3. Scanning electron micrographs and results of SEM-EDX analysis (arrows) of biofilm samples from MJB (A and C) and RTB (B and D). Bar, 10 µm.

filaments or the sheaths of bacteria in the MJB sample (Fig. 3C),whereas generally spheroid or amorphous forms of ferrihydriteconnected to microorganisms in the RTB sample (Fig. 3D).Therefore, it can be supposed that the observed microorgan-isms in both sites contribute actively and/or passively to themineral precipitations of iron hydroxides as described by Kon-hauser (1998). A similar phenomenon was also described byBarton et al. (2007) in the case of a rock sample from calcare-ous siltstones within Carlsbad Cavern, New Mexico, USA. It isinteresting to note that in the case of the RTB sample, the pres-ence of arsenic was also detected in 2.25 weight%. Casanovaet al. (1999) also recognized similar active arsenic concentrationby ferruginous bacteria in the Cezallier hydrothermal springs(Massif Central, France).

Clone Library AnalysisA total of 173 and 177 clones were analyzed to reveal the bac-

terial diversity of the biofilms developed on the walls of MolnarJanos cave (MJB) and Rudas Torok spring cave (RTB), respec-tively. Rarefaction analysis was applied to evaluate whether the

screening of the clones was satisfactory to estimate diversitywithin the clone libraries. Rarefaction curves (Fig. 4) representthe number of processed clones against the number of differentARDRA patterns (34 and 45) detected in the MJB and RTBclone libraries, respectively. The calculated rarefaction curvesdid not reach asymptotes, indicating that the analysis of an in-creasing number of clones would have revealed further diversity.This is in agreement with the results of previous reports, whichalso presumed high diversity of cave wall-colonizing bacteriaon the basis of rarefaction analysis (Pasic et al. 2010; Porteret al. 2009; Portillo et al. 2009).

Sequence analysis of the MJB and RTB clone sequences re-vealed 15 and 14 different phylogenetic lineages within the do-main Bacteria (Fig. 5) in accordance with the nearest describedspecies. Recently, a similarly broad distribution of bacterial phy-logenetic lineages was retrieved from other cave environmentsas shown in Table 1. In comparison with the results obtained bythe phylogenetic analysis of bacterial communities associatedwith cave environments, the members of the phyla Proteobac-teria, Bacteroidetes, Actinobacteria and Planctomycetes weredetected most frequently. Clones and/or isolates belonging to

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616 A. K. BORSODI ET AL.

FIG. 4. Rarefaction curves for the different ARDRA patterns of 16S rDNA clones from the Molnar Janos cave (MJB) and the Rudas Torok spring cave (RTB).

Gammaproteobacteria were identified from each cave sampleincluding MJB and RTB samples, as well.

Although the MJB and RTB clone libraries shared 12 di-visions, the distribution of the clones greatly differed amongthem. The most abundant group of clone sequences (Fig. 5)was affiliated with Firmicutes in the MJB library (40%) andDeltaproteobacteria in the RTB library (44%). Other cultivation-independent investigations on microbial communities associ-ated with cave environments (Table 1) also showed that se-quences affiliated with Proteobacteria constituted the most abun-dant detected fraction (Barton et al. 2007; Macalady et al.

2006; Pasic et al. 2010; Porter et al. 2009; Portillo et al. 2009;Schabereiter-Gurtner et al. 2002; Zhou et al. 2007).

Contrary to other results, relatively few 16S rDNA cloneswere affiliated with Gammaproteobacteria (4% of MJB and 8%of RTB) in both clone libraries. At the same time, the domi-nance of Deltaproteobacteria within the phylum Proteobacteria,which was detected in the RTB sample, seems to be a uniquephenomenon, as no similar finding has been published fromother karstic cave habitats so far. In agreement with a cave studyby Pasic et al. (2010), the absence of Epsilonproteobacteria wasalso observed in both of our clone libraries.

FIG. 5. Phylogenetic distribution of 16S rDNA sequences of the clone libraries from the Molnar Janos cave (MJB) and the Rudas Torok spring cave (RTB).

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TABLE 1Comparison of bacterial phylogenetic divisions detected from different cave environments

Aqu

ifica

e

The

rmod

esul

foba

cter

ia

Chl

orofl

exi

Nitr

ospi

rae

Cya

noba

cter

ia

Chl

orob

i

Alp

hapr

oteo

bact

eria

Bet

apro

teob

acte

ria

Gam

map

rote

obac

teri

a

Del

tapr

oteo

bact

eria

Eps

lonp

rote

obac

teri

a

Firm

icut

es

Act

inob

acte

ria

Plan

ctom

ycet

es

Aci

doba

cter

ia

Bac

tero

idet

es

Ver

ruco

mic

robi

a

Gem

mat

imon

adet

es

Oth

er

Molnar Janos Cave, Hungary (this study) + + + + + + + + + + + + + + +Rudas-Torok Bath, Hungary (this study) + + + + + + + + + + + + + +Pajsarjeva jama, Sloveniaa + + + + + + + + + + +Movile Cave, Romaniab + + + + + + + + + + +Frasassi Caves, Italyb,c,d,e + + + + + + + + + + + +Altamira Cave, Spainf + + + + + + + + + + + + +Tito Bustillo Cave, Spaing + + + + + + + +Lechuguilla and Spider Caves, New Mexico, USAh + + + + + + +Carlsbad Cavern, New Mexico, USAi + + + + + + +Wind Cave, South Dakota, USAj + + + + + + + + + + + +Cesspool Cave, Virginia, USAb,k + + + + + +Lower Kane Cave, Wyoming, USAb + + + + +Nullarbor Caves, Australial + + + + + + + +Niu Cave, Chinam + + + + + + + + + + +

Data from: aPasic et al. 2009; bPorter et al. 2009; cVlasceanu et al. 2000; dMacalady et al. 2006; eMacalady et al. 2007; fPortillo et al. 2009;gSchabereiter-Gurtner et al. 2002; hNorthup et al. 2003; iBarton et al. 2007; jChelius and Moore 2004; kEngel et al. 2001; lHolmes et al. 2001;mZhou et al. 2007.

Until now, high numbers of clones related to Firmicutes (LowG+C Gram-positives), found in the MJB sample, has not beendescribed from other deep karst systems. Among the Gram-positive bacteria, representatives of Actinobacteria (High G+CGram-positives) were detected as dominant members of themicrobial communities in the case of an iron-rich limestone cavewithin Carlsbad Cavern, New Mexico, USA (Barton et al. 2007).In both of our clone libraries, the second largest fraction (Fig. 4)was related to Actinobacteria (11% of MJB and 12% of RTB).

In addition, 11% of the clones represented Chloroflexi inthe RTB library. Chloroflexi (green non-sulfur bacteria) relatedclones were also frequently found in other cave environments(Barton et al. 2007; Chelius and Moore 2004; Pasic et al. 2010;Portillo et al. 2009; Schabereiter-Gurtner et al. 2002; Zhouet al. 2007). Other clone sequences (Thermodesulfobacteria,Nitrospirae, Chlorobi, Planctomycetes, Bacteroidetes and Ver-rucomicrobia) represented minor fractions (<10%) in libraries(Fig. 5). Nevertheless, it is interesting to note that phylotypes af-filiated with the deeply branching Thermodesulfobacteria havenot been reported from deep karst caves so far. Contrary tothe relatively low proportion of phylotypes related to Nitro-spirae, Planctomycetes and Bacteroidetes, the occurrence oftheir representatives seems to be constant in cave environments(Table 1).

Phylotypes affiliated with Aquificeae, Acidobacteria andGemmatimonadetes were unique to the MJB library, andCyanobacteria and Alphaproteobacteria to the RTB library(Fig. 5). Despite the relatively few cultivated species, and onthe basis of cultivation-independent phylogenetic analysis, Aci-dobacteria (with at least eight subdivisions) is thought to be as di-verse as the phylum Proteobacteria (Ludwig et al. 1997). In con-trast with our finding, Acidobacteria-related phylotypes formedabundant fractions in clone libraries from the Tito Bustillo Cave,Spain (Schabereiter-Gurtner et al. 2002), the Wind Cave, SouthDakota, USA (Chelius and Moore 2004) and the Niu Cave,China (Zhou et al. 2007). At the same time, representativesof Aquificeae, Cyanobacteria and Gemmatimonadetes were de-tected only sporadically in other caves (Barton et al. 2007; Pasicet al. 2010; Portillo et al. 2009; Zhou et al. 2007).

The nearest published relative of almost all clones from theMJB and RTB libraries was most closely related to unculturedbacterium clone sequences according to the BLAST search re-sult (Tables 2 and 3). Similarity values to 16S rDNA sequencesrecorded in the EMBL database ranged from 82% to 98%. In theinvestigated libraries, several clones were most closely affiliatedwith uncultured clone sequences originating from similar caveand subsurface environments (e.g., Frasassi Cave System, Italy;Cesspool Cave, USA; Wind Cave, USA; subsurface geothermal

Dow

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and

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ity],

[D

r A

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at 0

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2

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91(4

56/5

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roba

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(AY

2689

36)

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foba

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JB-1

0(F

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82(3

45/4

18)

uncu

lture

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ifor

nia,

USA

81(3

78/4

66)

Geo

ther

mob

acte

rium

ferr

ired

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s(A

F411

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(FR

7544

57)

395

(443

/466

)un

cultu

red

bact

eriu

mcl

one

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1511

2(A

M99

7775

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uth-

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an,

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nea

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(354

/443

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bact

eriu

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drog

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hilu

m(A

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514)

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exi

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-86

(FR

7544

84)

384

(414

/489

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cultu

red

Chl

orofl

exi

bact

eriu

mcl

one

CB

1893

b.64

(EF0

7616

3)

wat

erat

dept

hof

428

m,

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ticO

cean

,Can

ada

Bas

in

80(3

44/4

32)

Ros

eifle

xus

cast

enho

bii

(AB

0412

26)

MJB

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(FR

7544

82)

183

(385

/461

)un

cultu

red

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mcl

one

2018

(EF1

8868

0)A

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ain

80(3

61/4

51)

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4483

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91(4

40/4

82)

uncu

lture

dba

cter

ium

clon

e18

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(EF4

5982

7)B

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sedi

men

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(376

/451

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9439

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eM

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R75

4480

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86(4

45/5

14)

uncu

lture

dba

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(EU

4911

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Peak

84(4

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94)

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tes

Nit

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(EU

0848

79)

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(FR

7544

73)

296

(474

/493

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cultu

red

Nit

rosp

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bact

eriu

mcl

one

HA

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(AB

1136

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95(4

68/4

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8255

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84(3

99/4

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54(A

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82(3

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90(4

47/4

92)

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lture

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ium

cont

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ated

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urfa

cese

dim

ent

core

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80(3

91/4

87)

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dith

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cill

usfe

rroo

xida

ns(C

P001

219)

MJB

-68

(FR

7544

79)

191

(410

/447

)un

cultu

red

bact

eriu

mcl

one

OR

CA

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21(D

Q82

3229

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aves

Nat

iona

lM

onum

ent,

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91(4

03/4

40)

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r A

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5:56

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Del

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eria

MJB

-12

(FR

7544

58)

986

(394

/457

)D

esul

fati

feru

laol

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vora

nsst

rain

LM

2801

(DQ

8267

24)

84(3

98/4

70)

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ulfa

tife

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nivo

rans

(DQ

8267

24)

MJB

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(FR

7544

78)

388

(451

/511

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cultu

red

bact

eriu

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pLW

-92

(DQ

0669

83)

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ngto

n,U

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(369

/473

)D

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JB-5

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4476

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96(4

28/4

43)

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(385

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8239

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rmic

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-26

(FR

7544

65)

1787

(406

/465

)un

cultu

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bact

eriu

mcl

one

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76(D

Q49

9315

)m

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lm,

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78(3

82/4

90)

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(AF3

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(FR

7544

68)

1291

(482

/528

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cultu

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(EU

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(406

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s(A

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49/5

01)

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79(3

91/4

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(FR

7544

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(420

/466

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cultu

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t,W

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81(4

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obac

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7)

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(FR

7544

63)

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(395

/446

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cultu

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885

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(440

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(FR

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(425

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(FR

7544

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(413

/463

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cultu

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(FR

7544

75)

694

(487

/518

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cultu

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(EU

3351

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(408

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(FR

7544

71)

186

(416

/480

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cultu

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0563

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3371

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in

77(3

59/4

68)

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342)

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(FR

7544

74)

189

(459

/515

)un

cultu

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eriu

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C-s

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(EU

3351

77)

satu

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aggr

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(385

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2

TAB

LE

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quen

ces

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sam

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nar

Jano

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grou

psan

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em

atch

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enet

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tions

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rest

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(%)∗∗

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ctom

ycet

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9(F

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4481

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91(4

01/4

37)

uncu

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94(4

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89)

uncu

lture

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82(3

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49)

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cter

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(FR

7544

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194

(484

/512

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84(3

96/4

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194

(471

/496

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r A

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5:56

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TAB

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RT

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bial

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RT

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4451

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RT

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RT

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96(4

62/4

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RT

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623

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r A

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18

June

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2

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LE

3A

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BIOFILM BACTERIAL COMMUNITIES 625

water, Japan; subsurface sediment, USA Altamira Cave, Spain)(Barns et al. 2007; Chelius and Moore 2004; Hirayama et al.2005; Macalady et al. 2007; Porter et al. 2009; Portillo et al.2009). In agreement with previous studies (Chelius and Moore2004; Pasic et al. 2010; Zhou et al. 2007), neither of the clonesfrom MJB and RTB samples was ecosystem-specific (charac-teristic only to biofilms developed on the walls of karst caves).

To attempt to understand the potential role of microorgan-isms inhabiting the Molnar Janos cave (MJB) and the Rudas-Torok spring cave (RTB), the sequences from both libraries werealigned against sequences of described bacterial species withknown metabolism (Tables 2 and 3). In almost all cases, the sim-ilarity values of the partial 16S rDNA clone sequences were be-low the level of species identification (>97%) (Stackebrandt andGoebel 1994). The low similarity to described species indicatedthat the MJB and RTB biofilm samples could harbor membersof hitherto unknown bacterial species. Microorganisms living inthermal caves are known to adapt to the special physio-chemistry(e.g., temperature, pH) and the frequently oligotrophic environ-ments. Nevertheless, since 2000 several new bacterial species(mainly Actinobacteria) have been described from caves andsubterranean environments (Jurado et al. 2005, 2009, 2010).

The knowledge of the metabolic properties and environmen-tal tolerance of closely related species could help us to under-stand the possible role of bacteria detected from caves. Accord-ing to the closely related species, both of the studied biofilmscould be characterized by phylogenetically diverse microbialcommunities. Although most of the MJB and RTB phylotypeswere found only in one of the two samples, common taxa werealso found in almost all phyla, including the genera of Geother-mobacterium (Thermodesulfobacteria), Levilinea (Chloroflexi),Nitrospira (Nitrospirae), Ignavibacterium (Chlorobi), Desul-fovibrio and Anaeromyxobacter (Deltaproteobacteria), Ther-molithobacter (Firmicutes), Acidothermus and Lentzea (Acti-nobacteria), Cytophaga (Bacteroidetes) and Methylacidiphilum(Verrucomicrobia).

Aside from the phylotypes related to the anaerobic andthermophilic species of Aquificeae, Thermodesulfobacteria andChloroflexi, several other clones in the MJB sample were af-filiated with aerobic or anaerobic thermophilic species of thegenera Thermaerobacter, Thermolithobacter and Thermoanaer-obacter within the phylum Firmicutes, and aerobic thermoaci-dophilic species of the genera Acidimicrobium and Acidother-mus within the phylum Actinobacteria (Table 2). The ratio ofclones related to thermophilic or hyperthermophilic bacterialspecies (e.g., Geothermobacter, Thermolithobacter, Acidother-mus) was lower (Table 3) in the case of the RTB sample, with theexception of Thermodesulfobacteria and Chloroflexi commonto the MJB sample.

It can be hypothesized on the basis of our results that the rela-tively high proportion of the indigenous MJB and RTB bacteriamay be due to the ascending hydrothermal waters related to thestudied caves. The higher proportion of thermophilic bacteriain the MJB sample can be explained by the higher temperature

(up to 70◦C) of the ascending hydrothermal waters character-istic of the Rose Hill area and connected to the Molnar Janoscave. Due to circumneutral pH, only a low abundance of phylo-types related to acidophilic bacteria was found in both biofilmsamples.

In addition to the phylogenetic heterogeneity found in oursamples, a large variety of metabolic types can also be presumed,including aerobic and anaerobic, chemolithotrophic autotrophicand chemoorganotrophic heterotrophic bacteria.

Several phylotypes from the MJB and RTB samples wererelated to bacterial species involved either in aerobic iron(FeII)-oxidizing (e.g., Acidithiobacillus ferrooxidans, Acidimicro-bium ferrooxidans, Ferrithrix thermotolerans) or anaerobiciron(FeIII)-reducing (e.g., Geothermobacterium ferrireducens,Thermolithobacter ferrireducens) metabolism (Tables 2 and 3).The phylogenetic analysis was also supported by the electronmicrographs of the biofilm samples and the results of SEM-EDXanalysis, confirming that iron metabolizing chemoautotrophicbacteria may have great ecological significance in these caveecosystems. A similar phenomenon was observed by Northupet al. (2000, 2003) and Barton et al. (2007).

Bacteria that can participate in the sulfur cycle appear tobe one of the most dominant group of cave microbial com-munities as reported in previous studies (Chelius and Moore2004; Engel et al. 2001; Macalady et al. 2006). In both ofour samples, a number of phylotypes were associated withbacterial species taking part in dissimilatory sulfate reductionprocesses either by lithotrophic (e.g., Thermodesulfobacteriumhydrogeniphilum, Ammonifex thiophilus) or by organotrophic(e.g., Desulfatibacillum alkenivorans, Desulfatiferula olefinivo-rans, Desulfuromonas michiganensis, Desulfovibrio africanus)metabolism.

The occurrence of these bacteria could be connected to therelatively high sulfate concentration (MJB 134 mg l−1, RTB333 mg l−1) measured in the waters of both sampling sites.However, mainly in the RTB sample, clones closely relatedto sulfur-oxidizing Gammaproteobacteria (e.g., Thiothrix unzii)were also found. In other 16S rDNA based molecular analyses,Thiothrix-related clone sequences were also identified in matand biofilm samples from the Sulphur River of Parker Cave,Kentucky, USA (Angert et al. 1998), Cesspool Cave, Virginia,USA (Engel et al. 2001) and a sulfidic spring outflow in theFrasassi Cave System, Italy (Macalady et al. 2006), as well assulfurous wells of cavernous limestone aquifers in the South-Western part of Hungary (Miseta et al. 2012).

In addition to aerobic chemoautotrophic sulfur-oxidizingbacteria, scarce occurrence of phylotypes related to anaerobicphototrophic bacteria (e.g. Allochromatium vinosum, Thiorho-dospira sibirica) and Cyanobacteria were only detected in theRTB sample where light penetrates occasionally (mainly duringthe sampling). The lack of phototrophic bacteria in the MJBsample can be the consequence of the fully phreatic conditions.

In addition, Nitrospirae-related obligate chemolithoau-totrophic nitrite oxidizing bacteria were detected recently from

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626 A. K. BORSODI ET AL.

several caves (Holmes et al. 2001; Northup et al. 2003; Pasicet al. 2010) including the MJB and RTB samples.

The presence of bacteria with chemoorganotrophicmetabolism in these oligotrophic environments depends primar-ily on the amount of allochthonous and autochthonous organicmatter. Previous, mainly cultivation-based studies of microbiotaassociated with cave environments, supported the occurrence ofchemoorganotrophic bacteria belonging to Actinobacteria andBacteroidetes (Canaveras et al. 2001; Engel et al. 2001; Grothet al. 2001). According to our cultivation-independent inves-tigation, not only clones related to the widely distributed andmetabolically versatile aerobic chemoorganotrophic species ofActinobacteria and Bacteroidetes, but also anaerobic sulfate-,sulfur- and nitrate-reducing Deltaproteobacteria were detectedin both the MJB and RTB samples.

The close physical interactions observed by SEM and thedetected phylogenetic and the revealed possible metabolic di-versity of bacteria indicate unimaginably complex and man-ifold interactions among the microorganisms participating inthe biofilm formation on the cave walls of the MJB and RTBsamples.

In conclusion, our results showed that SEM and molecu-lar cloning investigations on the biofilms of two cave walls inthe Buda Thermal Karst System (Hungary) uncovered morpho-logically and phylogenetically diverse microbial communities.The distribution of clones was markedly different between thestudied areas, inasmuch as the highest number of clones wasaffiliated with Firmicutes in the MJB, and with Deltaproteobac-teria in the RTB. The results of the phylogenetic analysis suggestthe existence of large diversity of mesophilic and thermophilicbacteria involved in the local sulfur and iron cycles at bothlocations. Studying the so far unknown bacterial assemblageshas revealed greater phylogenetic diversity than previously de-scribed from other cave environments.

This work represents only the first step toward the explorationof microbial diversity of BTKS and has drawn our attention tothe necessary of a more detailed investigation on microbial di-versity with respect to Archaea, as well. Further expanded moni-toring of biofilms by applying a polyphasic approach, involvingboth cultivation-based and cultivation-independent (DNA- aswell as RNA-based) techniques, may allow a better understand-ing of the impact of these microorganisms on cave formation.

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