Biofilm Bacterial Communities Inhabiting the Cave Walls of...
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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: [email protected]
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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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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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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(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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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
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
TAB
LE
2A
ssig
nmen
tof
clon
ese
quen
ces
ofbi
ofilm
sam
ples
from
the
Mol
nar
Jano
sca
ve(M
JB)
tota
xono
mic
grou
psan
dth
ecl
oses
tseq
uenc
em
atch
esof
know
nph
ylog
enet
icaf
filia
tions
Rep
rese
ntat
ive
No.
ofSi
mila
rity
Nea
rest
publ
ishe
dE
nvir
onm
enta
lSi
mila
rity
Nea
rest
desc
ribe
dsp
ecie
scl
one∗
clon
es(%
)∗∗re
lativ
eby
BL
AST
∗so
urce
(%)∗∗
byE
zTax
on∗
Aqu
ifica
eM
JB-1
3(F
R75
4459
)6
91(4
56/5
00)
uncu
lture
dba
cter
ium
clon
eF1
32X
2(G
Q26
2856
)si
mul
ated
low
leve
lwas
tesi
te,I
daho
Nat
iona
lL
abs,
USA
81(4
03/4
94)
Des
ulfu
roba
cter
ium
paci
ficum
(AY
2689
36)
The
rmod
esul
foba
cter
iaM
JB-1
0(F
R75
4456
)9
82(3
45/4
18)
uncu
lture
dba
cter
ium
clon
eFC
PU45
3(E
F515
964)
gras
slan
dso
il,N
orth
ern
Cal
ifor
nia,
USA
81(3
78/4
66)
Geo
ther
mob
acte
rium
ferr
ired
ucen
s(A
F411
013)
MJB
-11
(FR
7544
57)
395
(443
/466
)un
cultu
red
bact
eriu
mcl
one
Ucb
1511
2(A
M99
7775
)So
uth-
Atla
ntic
Oce
an,
Gui
nea
Bas
in80
(354
/443
)T
herm
odes
ulfo
bact
eriu
mhy
drog
enip
hilu
m(A
F332
514)
Chl
orofl
exi
MJB
-86
(FR
7544
84)
384
(414
/489
)un
cultu
red
Chl
orofl
exi
bact
eriu
mcl
one
CB
1893
b.64
(EF0
7616
3)
wat
erat
dept
hof
428
m,
Arc
ticO
cean
,Can
ada
Bas
in
80(3
44/4
32)
Ros
eifle
xus
cast
enho
bii
(AB
0412
26)
MJB
-80
(FR
7544
82)
183
(385
/461
)un
cultu
red
bact
eriu
mcl
one
2018
(EF1
8868
0)A
ltam
ira
Cav
e,Sp
ain
80(3
61/4
51)
Spha
erob
acte
rth
erm
ophi
lus
(CP0
0182
4)M
JB-8
5(F
R75
4483
)1
91(4
40/4
82)
uncu
lture
dba
cter
ium
clon
e18
1b1
(EF4
5982
7)B
altic
Sea
sedi
men
t83
(376
/451
)L
evil
inea
sacc
haro
lyti
ca(A
B10
9439
)N
itros
pira
eM
JB-6
9(F
R75
4480
)6
86(4
45/5
14)
uncu
lture
dba
cter
ium
clon
eP9
X2b
7D09
(EU
4911
91)
seafl
oor
lava
sfr
omth
eL
oi’h
iSea
mou
ntPi
sces
Peak
84(4
16/4
94)
Can
dida
tes
Nit
rosp
ira
bock
iana
(EU
0848
79)
MJB
-45
(FR
7544
73)
296
(474
/493
)un
cultu
red
Nit
rosp
ira
bact
eriu
mcl
one
HA
uD-L
B38
(AB
1136
21)
95(4
68/4
91)
Nit
rosp
ira
mos
covi
ensi
s(X
8255
8)
Chl
orob
iM
JB-3
7(F
R75
4470
)5
84(3
99/4
77)
uncu
lture
dba
cter
ium
clon
ePK
54(A
Y55
5788
)B
orK
hlue
ngho
tspr
ing,
Tha
iland
82(3
86/4
72)
Igna
viba
cter
ium
albu
m(A
B47
8415
)B
etap
rote
obac
teri
aM
JB-4
4(F
R75
4472
)1
94(4
71/4
99)
uncu
lture
dba
cter
ium
clon
em
le1-
7(A
F280
846)
bior
eact
ortr
eatin
gph
arm
aceu
tical
was
tew
ater
89(4
30/4
83)
Den
itra
tiso
ma
oest
radi
olic
um(A
Y87
9297
)
Gam
map
rote
obac
teri
aM
JB-2
4(F
R75
4464
)6
90(4
47/4
92)
uncu
lture
dba
cter
ium
bact
eriu
mcl
one
T10
.003
3(E
F457
309)
uran
ium
cont
amin
ated
subs
urfa
cese
dim
ent
core
,USA
80(3
91/4
87)
Aci
dith
ioba
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
-17N
21(D
Q82
3229
)O
rego
nC
aves
Nat
iona
lM
onum
ent,
USA
91(4
03/4
40)
Met
hylo
cald
umsz
eged
iens
e(U
8930
0)
618
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
Del
tapr
oteo
bact
eria
MJB
-12
(FR
7544
58)
986
(394
/457
)D
esul
fati
feru
laol
efini
vora
nsst
rain
LM
2801
(DQ
8267
24)
84(3
98/4
70)
Des
ulfa
tife
rula
olefi
nivo
rans
(DQ
8267
24)
MJB
-59
(FR
7544
78)
388
(451
/511
)un
cultu
red
bact
eriu
mcl
one
pLW
-92
(DQ
0669
83)
sedi
men
tof
Lak
eW
ashi
ngto
n,U
SA78
(369
/473
)D
esul
fovi
brio
afri
canu
s(X
9923
6)M
JB-5
5(F
R75
4476
)1
96(4
28/4
43)
unid
entifi
edba
cter
ium
clon
eN
eu2P
1-42
(AJ5
1832
0)se
dim
ento
fa
rese
rvoi
r,Sa
xony
,Ger
man
y78
(385
/496
)A
naer
omyx
obac
ter
deha
loge
nans
(AF3
8239
6)Fi
rmic
utes
MJB
-26
(FR
7544
65)
1787
(406
/465
)un
cultu
red
bact
eriu
mcl
one
CV
76(D
Q49
9315
)m
icro
bial
biofi
lm,
Fras
assi
cave
syst
em,
Ital
y
78(3
82/4
90)
The
rmae
roba
cter
subt
erra
neus
(AF3
4356
6)
MJB
-32
(FR
7544
68)
1291
(482
/528
)un
cultu
red
bact
eriu
mcl
one
Bac
C-s
053
(EU
3351
81)
satu
rate
dC
hori
zon
soil
aggr
egat
e82
(406
/493
)T
herm
olit
hoba
cter
ferr
ired
ucen
s(A
F282
253)
MJB
-2(F
R75
4452
)11
89(4
49/5
01)
unid
entifi
edba
cter
ium
clon
eFI
-2M
D11
(EF2
2068
7)so
ilen
viro
nmen
tsun
der
Em
petr
umru
brum
,Fa
lkla
ndIs
land
s
79(3
91/4
91)
The
rmae
roba
cter
naga
saki
ensi
s(A
B06
1441
)
MJB
-27
(FR
7544
66)
990
(420
/466
)un
cultu
red
bact
eriu
mcl
one
WC
B15
4(A
Y21
7537
)sa
tura
ted
sedi
men
t,W
ind
Cav
e,So
uth
Dak
ota,
USA
81(4
06/5
02)
The
rmoa
naer
obac
ter
ther
moc
opri
ae(L
0916
7)
MJB
-20
(FR
7544
63)
888
(395
/446
)un
cultu
red
bact
eriu
mcl
one
885
(EF1
8831
2)A
ltam
ira
Cav
e,Sp
ain
80(3
41/4
28)
Am
mon
ifex
thio
phil
us(E
F554
597)
MJB
-5(F
R75
4454
)6
90(4
60/5
08)
unid
entifi
edba
cter
ium
clon
eFI
-2M
D11
(EF2
2068
7)so
ilen
viro
nmen
tsun
der
Em
petr
umru
brum
,Fa
lkla
ndIs
land
s
80(3
99/4
97)
The
rmae
roba
cter
lito
rali
s(A
Y93
6496
)
MJB
-15
(FR
7544
61)
595
(440
/462
)un
cultu
red
bact
eriu
mcl
one
tpb-
16-A
B-G
09(D
Q40
7307
)gr
ound
wat
er,F
ield
Res
earc
hC
ente
r,O
akR
idge
,USA
76(3
63/4
78)
Terr
ibac
illu
ssa
ccha
roph
ilus
(AB
2438
45)
MJB
-29
(FR
7544
67)
188
(425
/482
)un
cultu
red
bact
eriu
mcl
one
HA
uD-M
B19
(AB
1136
01)
subs
urfa
cege
othe
rmal
wat
erof
ago
ldm
ine,
Japa
n
79(3
72/4
72)
The
rmoa
naer
obac
ter
pseu
deth
anol
icus
(CP0
0092
4)
Act
inob
acte
ria
MJB
-19
(FR
7544
62)
1189
(413
/463
)un
cultu
red
bact
eriu
mcl
one
CV
76(D
Q49
9315
)m
icro
bial
biofi
lm,
Fras
assi
cave
syst
em,
Ital
y
83(3
75/4
50)
Aci
dim
icro
bium
ferr
ooxi
dans
(CP0
0163
1)
MJB
-50
(FR
7544
75)
694
(487
/518
)un
cultu
red
bact
eriu
mcl
one
Bac
C-s
046
(EU
3351
92)
satu
rate
dC
hori
zon
soil
aggr
egat
e84
(408
/487
)A
cido
ther
mus
cell
ulol
ytic
us(C
P000
481)
MJB
-40
(FR
7544
71)
186
(416
/480
)un
cultu
red
bact
eriu
mcl
one
CB
0563
b.90
(GQ
3371
19)
wat
erat
dept
hof
1000
m,
Arc
ticO
cean
,Can
ada
Bas
in
77(3
59/4
68)
Lei
fson
iapo
ae(A
F116
342)
MJB
-46
(FR
7544
74)
189
(459
/515
)un
cultu
red
bact
eriu
mcl
one
Bac
C-s
021
(EU
3351
77)
satu
rate
dC
hori
zon
soil
aggr
egat
e79
(385
/487
)L
entz
eaca
lifo
rnie
nsis
(AF1
7443
5)(C
onti
nued
onne
xtpa
ge)
619
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
TAB
LE
2A
ssig
nmen
tof
clon
ese
quen
ces
ofbi
ofilm
sam
ples
from
the
Mol
nar
Jano
sca
ve(M
JB)
tota
xono
mic
grou
psan
dth
ecl
oses
tseq
uenc
em
atch
esof
know
nph
ylog
enet
icaf
filia
tions
(Con
tinu
ed)
Rep
rese
ntat
ive
No.
ofSi
mila
rity
Nea
rest
publ
ishe
dE
nvir
onm
enta
lSi
mila
rity
Nea
rest
desc
ribe
dsp
ecie
scl
one∗
clon
es(%
)∗∗re
lativ
eby
BL
AST
∗so
urce
(%)∗∗
byE
zTax
on∗
Plan
ctom
ycet
esM
JB-7
9(F
R75
4481
)2
91(4
01/4
37)
uncu
lture
dpl
anct
omyc
ete
clon
e49
S12B
12(D
Q83
7238
)co
asta
laqu
ifer
,Don
ana
Nat
iona
lPar
k,Sp
ain
83(3
60/4
34)
Can
dida
tes
Bro
cadi
aan
amm
oxid
ans
(AF3
7599
4)A
cido
bact
eria
MJB
-9(F
R75
4455
)6
94(4
61/4
89)
uncu
lture
dA
cido
bact
eria
bact
eriu
mcl
one
RP7
.003
0(E
F457
501)
uran
ium
cont
amin
ated
subs
urfa
cese
dim
ent
core
,USA
82(3
70/4
49)
Aci
doba
cter
ium
caps
ulat
um(D
2617
1)
MJB
-34
(FR
7544
69)
194
(484
/512
)un
iden
tified
bact
eriu
mcl
one
47C
1B
UL
KT
7s(E
F605
742)
bulk
soil
from
form
erar
able
field
colo
nize
dby
wee
ds
84(3
96/4
73)
Can
dida
tes
Chl
orac
idob
acte
rium
ther
mop
hilu
m(E
F531
33S
MJB
-5S
(FR
7544
77)
194
(471
/496
)un
cultu
red
Aci
doba
cter
iaba
cter
ium
clon
eR
3.00
48(E
F457
449)
83(3
92/4
71)
Can
dida
tes
Kor
ibac
ter
vers
atil
e(C
P000
360)
Bac
tero
idet
esM
JB-4
(FR
7544
53)
489
(391
/438
)un
cultu
red
bact
eriu
mcl
one
DO
KN
OFE
RT
clon
e636
(DQ
8295
49)
agri
cultu
rals
oil,
Switz
erla
nd80
(392
/490
)C
ytop
haga
hutc
hins
onii
(CP0
0038
3)
Ver
ruco
mic
robi
aM
JB-S
9(F
R75
4485
)5
88(4
64/5
27)
uncu
lture
dba
cter
ium
clon
eFC
116
S98
(EU
6625
08)
mic
robi
alm
atfr
omsu
lfidi
cca
vest
ream
,Fr
asas
siC
aves
,Ita
ly
79(3
90/4
93)
Met
hyla
cidi
phil
umin
fern
orum
(CP0
0097
5)
Gem
mat
imon
adet
esM
JB-1
4(F
R75
4460
)9
93(4
71/5
03)
unid
entifi
edba
cter
ium
clon
eM
1B
ulk
T7s
42(E
F605
653)
bulk
soil
from
agri
cultu
rabl
efie
ldcr
oppe
dw
ithm
aize
83(3
69/4
59)
Gem
mat
imon
asau
rant
iaca
(AB
0727
35)
∗ Num
ber
inpa
rent
hesi
sin
dica
tes
the
Gen
Ban
kac
cess
ion
num
ber.
∗∗N
umbe
rin
pare
nthe
sis
indi
cate
sth
ese
quen
cele
ngth
used
for
calc
ulat
ion
ofse
quen
cesi
mila
rity
.
620
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
TAB
LE
3A
ssig
nmen
tof
clon
ese
quen
ces
ofbi
ofilm
sam
ples
from
the
Rud
as-T
orok
spri
ngca
ve(R
TB
)to
taxo
nom
icgr
oups
and
the
clos
ests
eque
nce
mat
ches
ofkn
own
phyl
ogen
etic
affil
iatio
ns
Rep
rese
ntat
ive
No.
ofSi
mila
rity
Nea
rest
publ
ishe
dE
nvir
onm
enta
lSi
mila
rity
Nea
rest
desc
ribe
dsp
ecie
scl
one∗
clon
es(%
)∗∗re
lativ
eby
BL
AST
∗so
urce
(%)∗∗
byE
zTax
on∗
The
rmod
esul
foba
cter
iaR
TB
-66
(FR
7544
36)
594
(458
/483
)un
cultu
red
bact
eriu
mcl
one
Am
b16
S76
6(E
F018
386)
trem
blin
gas
pen
rhiz
osph
ere
81(3
83/4
70)
Geo
ther
mob
acte
rium
ferr
ired
ucen
s(A
F411
013)
Chl
orofl
exi
RT
B-6
(FR
7544
11)
1290
(436
/483
)un
cultu
red
bact
eriu
mcl
one
p26f
23ok
(FJ4
7920
4)ta
llgr
ass
prai
rie,
Okl
ahom
a,U
SA88
(393
/449
)B
elli
line
aca
ldifi
stul
ae(A
B24
3672
)R
TB
-17
(FR
7544
17)
796
(462
/480
)un
cultu
red
bact
eriu
mcl
one
TA3
17(E
U74
6695
)dr
inki
ngw
ater
syst
em,
Tri
kala
City
,Gre
ece
88(4
11/4
65)
Ana
erol
inea
ther
mop
hila
(AB
0464
13)
RT
B-5
4(F
R75
4433
)1
96(4
18/4
31)
uncu
lture
dba
cter
ium
clon
ep3
6l20
ok(F
J478
597)
tall
gras
spr
airi
e,O
klah
oma,
USA
89(3
71/4
17)
Lev
ilin
easa
ccha
roly
tica
(AB
1094
39)
Nitr
ospi
rae
RT
B-3
4(F
R75
4423
)4
96(4
21/4
36)
uncu
lture
dN
itro
spir
asp
.cl
one
B8
(EU
8503
62)
was
tew
ater
trea
tmen
tpl
antE
lste
rwer
da,
Ger
man
y
88(4
16/4
71)
Nit
rosp
ira
mos
covi
ensi
s(X
8255
8)
RT
B-5
7(F
R75
4435
)1
98(4
37/4
44)
uncu
lture
dba
cter
ium
clon
eC
V82
(DQ
4993
18)
mic
robi
albi
ofilm
,Fr
asas
sica
vesy
stem
,It
aly
94(4
17/4
43)
Can
dida
tus
Nit
rosp
ira
bock
iana
(EU
0848
79)
Cya
noba
cter
iaR
TB
-5(F
R75
4410
)1
90(4
29/4
76)
uncu
lture
dba
cter
ium
clon
eoc
31(A
Y49
1574
)m
icro
bial
fuel
cell
79(3
49/4
44)
Pro
chlo
roco
ccus
mar
inus
(AF1
8096
7)C
hlor
obi
RT
B-6
9(F
R75
4438
)3
94(4
69/4
97)
uncu
lture
dba
cter
ium
clon
eK
as17
5B(E
F203
206)
sedi
men
t,L
ake
Kas
tori
a,G
reec
e91
(429
/473
)Ig
navi
bact
eriu
mal
bum
(AB
4784
15)
Alp
hapr
oteo
bact
eria
RT
B-5
6(F
R75
4434
)8
92(3
73/4
05)
uncu
lture
dba
cter
ium
clon
eY
E20
1C03
(FJ6
9454
6)fr
eshw
ater
,Yen
isey
Riv
er,
Rus
sia
79(3
47/4
37)
Bre
vund
imon
asal
ba(A
J227
785)
RT
B-5
1(F
R75
4431
)1
88(4
09/4
61)
uncu
lture
dR
hodo
plan
essp
.cl
one
AL
PHA
6A(A
Y49
4635
)
Salm
osa
lar
gill
89(3
91/4
36)
Sino
rhiz
obiu
mch
iapa
necu
m(E
U28
6550
)
Bet
apro
teob
acte
ria
RT
B-4
1(F
R75
4426
)1
96(4
70/4
88)
uncu
lture
dba
cter
ium
clon
eK
ZN
MV
-30-
B39
(FJ7
1260
9)
Kaz
anM
udV
olca
no,E
ast
Med
iterr
anea
nSe
a91
(417
/456
)D
echl
orom
onas
agit
ata
(AF0
4746
2)
(Con
tinu
edon
next
page
)
621
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
TAB
LE
3A
ssig
nmen
tof
clon
ese
quen
ces
ofbi
ofilm
sam
ples
from
the
Rud
as-T
orok
spri
ngca
ve(R
TB
)to
taxo
nom
icgr
oups
and
the
clos
ests
eque
nce
mat
ches
ofkn
own
phyl
ogen
etic
affil
iatio
ns(C
onti
nued
)
Rep
rese
ntat
ive
No.
ofSi
mila
rity
Nea
rest
publ
ishe
dE
nvir
onm
enta
lSi
mila
rity
Nea
rest
desc
ribe
dsp
ecie
scl
one∗
clon
es(%
)∗∗re
lativ
eby
BL
AST
∗so
urce
(%)∗∗
byE
zTax
on∗
Gam
map
rote
obac
teri
aR
TB
-8(F
R75
4412
)6
98(4
39/4
52)
uncu
lture
dba
cter
ium
clon
eC
C1
16S
8(E
U66
2402
)m
icro
bial
mat
ofC
essp
ool
Cav
e,V
irgi
nia,
USA
98(4
29/4
40)
Thi
othr
ixun
zii(
L79
961)
RT
B-1
11(F
R75
4451
)4
90(4
50/4
95)
uncu
lture
dT
hiot
rich
acea
eba
cter
ium
clon
eD
1044
(EU
2668
08)
tar-
oilc
onta
min
ated
aqui
fer
sedi
men
t87
(400
/459
)A
lloc
hrom
atiu
mvi
nosu
m(A
CQ
Q01
0000
50)
RT
B-4
(FR
7544
09)
391
(457
/498
)un
cultu
red
gam
ma
prot
eoba
cter
ium
clon
e3P
JM42
(FJ5
3507
5)
kars
ticca
vew
allb
iofil
m,
Slov
enia
90(4
34/4
82)
Thi
orho
dosp
ira
sibi
rica
(AJ0
0653
0)
RT
B-3
2(F
R75
4422
)1
96(4
62/4
81)
uncu
lture
dba
cter
ium
clon
eW
-Btb
759
(DQ
0179
20)
upla
ndst
ream
90(4
23/4
71)
Aqu
icel
lasi
phon
is(A
Y35
9283
)
Del
tapr
oteo
bact
eria
RT
B-2
8(F
R75
4420
)17
95(4
91/5
15)
uncu
lture
dba
cter
ium
clon
eA
V9-
172
(AM
1818
81)
prof
unda
lsed
imen
t,L
ake
Kin
nere
t,Is
rael
84(4
28/5
11)
Des
ulfa
tiba
cill
umal
keni
vora
ns(A
Y49
3562
)R
TB
-46
(FR
7544
28)
1486
(448
/520
)un
cultu
red
bact
eriu
mcl
one
BB
-HB
102
(GQ
8443
55)
was
tew
ater
trea
tmen
tpl
antb
iofil
m,C
hina
85(4
20/4
96)
Des
ulfu
rom
onas
mic
higa
nens
is(A
F357
915)
RT
B-7
9(F
R75
4441
)13
91(4
82/5
28)
uncu
lture
dba
cter
ium
clon
eB
B-H
B39
(GQ
8443
47)
was
tew
ater
trea
tmen
tpl
antb
iofil
m,C
hina
84(4
27/5
09)
Geo
bact
ersu
lfur
redu
cens
(AE
0171
80)
RT
B-2
2(F
R75
4418
)6
88(3
96/4
49)
uncu
lture
dde
ltapr
oteo
bact
eriu
mcl
one
JRC
BII
23(D
Q25
2377
)
jute
-ret
ting
wat
er83
(426
/513
)G
eoth
erm
obac
ter
ehrl
ichi
i(A
Y15
5599
)
RT
B-4
7(F
R75
4429
)6
93(4
74/5
06)
uncu
lture
dba
cter
ium
clon
eFF
CH
1147
3(E
U13
4344
)so
il,O
klah
oma,
USA
81(3
97/4
91)
Hip
pea
mar
itim
a(Y
1829
2)
RT
B-1
00(F
R75
4450
)5
85(3
74/4
38)
uncu
lture
dba
cter
ium
clon
e70
25P1
B15
(EF5
6203
9)de
epgr
aniti
cfr
acru
rew
ater
,Col
orad
o,U
SA82
(367
/445
)B
ilop
hila
wad
swor
thia
(AJ8
6704
9)R
TB
-14
(FR
7544
15)
489
(428
/476
)un
cultu
red
soil
bact
eriu
mcl
one
Bac
t.dry
.1A
CA
TD
02(G
U37
5335
)
oil-
field
soil
79(3
85/4
87)
Geo
alka
liba
cter
ferr
ihyd
riti
cus
(DQ
3093
26)
RT
B-5
0(F
R75
4430
)3
91(4
74/5
16)
uncu
lture
dba
cter
ium
clon
e70
25P4
B71
(EF5
6209
7)de
epgr
aniti
cfr
acru
rew
ater
,Col
orad
o,U
SA81
(389
/480
)St
igm
atel
lahy
brid
a(D
Q76
8129
)R
TB
-36
(FR
7544
24)
289
(466
/521
)un
cultu
red
bact
eriu
mcl
one
Kas
139B
(EF2
0318
8)se
dim
ent,
Lak
eK
asto
ria,
Gre
ece
81(4
07/5
04)
Des
ulfo
vibr
ioox
amic
us(D
Q12
2124
)R
TB
-85
(FR
7544
43)
289
(422
/469
)un
cultu
red
bact
eriu
mcl
one
JMY
B36
-32
(FJ8
1056
0)su
bsur
face
grou
ndw
ater
78(3
45/4
41)
Hya
lang
ium
min
utum
(DQ
7681
24)
RT
B-9
3(F
R75
4447
)2
93(4
51/4
83)
uncu
lture
dcl
one
DO
KC
ON
FYM
clon
e220
(DQ
8285
12)
agri
cultu
rals
oil,
Switz
erla
nd86
(408
/474
)H
alia
ngiu
mte
pidu
m(A
B06
2751
)
RT
B-3
8(F
R75
4425
)1
94(3
96/4
21)
uncu
lture
dso
ilba
cter
ium
clon
e10
74-1
(AY
3265
84)
fore
stso
il,W
este
rnA
maz
on81
(343
/421
)Pe
loba
cter
carb
inol
icus
(CP0
0014
2)
622
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
RT
B-1
3(F
R75
4414
)1
95(4
96/5
20)
uncu
lture
dba
cter
ium
clon
eM
ND
4(A
F293
008)
Gre
enB
ayfe
rrom
anga
nous
mic
rono
dule
84(4
26/5
08)
Ana
erom
yxob
acte
rde
halo
gena
ns(A
F382
396)
RT
B-6
7(F
R75
4437
)1
88(4
18/4
75)
uncu
lture
dpr
oteo
bact
eriu
mcl
one
DO
KB
IOD
YN
clon
e484
(DQ
8281
52)
agri
cultu
rals
oil,
Switz
erla
nd85
(410
/479
)D
esul
fovi
bro
afri
canu
s(E
U65
9693
)
RT
B-7
5(F
R75
4440
)1
96(4
59/4
75)
uncu
lture
dba
cter
ium
clon
eC
M41
(AM
9100
70)
rice
field
soil,
Han
gzho
u,C
hina
85(3
91/4
60)
Mel
itta
ngiu
mli
chen
icol
a(A
M93
0269
)Fi
rmic
utes
RT
B-8
4(F
R75
4442
)4
92(4
78/5
17)
uncu
lture
dba
cter
ium
clon
eIn
o-R
a3(F
J621
542)
fres
hwat
erdi
tch
sedi
men
t,O
oijp
olde
r,N
ethe
rlan
ds80
(406
/505
)T
herm
olit
hoba
cter
carb
oxyd
ivor
ans
(DQ
0958
62)
RT
B-1
0(F
R75
4413
)2
82(3
78/4
56)
uncu
lture
dba
cter
ium
clon
eD
14R
15C
34(F
M95
6742
)ri
cefie
ldso
il,H
angz
hou,
Chi
na77
(372
/484
)Vi
rgib
acil
lus
halo
phil
us(A
B24
3851
)R
TB
-27
(FR
7544
19)
291
(441
/482
)un
cultu
red
bact
eriu
mcl
one
PS-B
a22
(EU
3996
64)
phen
ol-d
egra
ding
slud
ge82
(388
/475
)C
arbo
xydo
ther
mus
side
roph
ilus
(EF5
4281
0)R
TB
-87
(FR
7544
45)
196
(439
/454
)un
cultu
red
bact
eriu
mcl
one
AK
YH
1182
(AY
9216
97)
farm
soil,
Min
neso
ta,
USA
82(3
82/4
63)
Hel
ioba
cill
usm
obil
is(A
B10
0835
)A
ctin
obac
teri
aR
TB
-31
(FR
7544
21)
888
(458
/518
)un
cultu
red
bact
eriu
mcl
one
Bac
C-s
021
(EU
3351
77)
satu
rate
dC
hori
zon
soil
aggr
egat
e78
(381
/488
)L
entz
eaca
lifo
rnie
nsis
(AF1
7443
5)R
TB
-43
(FR
7544
27)
586
(465
/539
)un
cultu
red
bact
eriu
mcl
one
Ba2
7(F
J640
812)
hydr
othe
rmal
vent
chim
ney,
Juan
deFu
caR
idge
78(3
79/4
83)
Aci
doth
erm
usce
llul
olyt
icus
(CP0
0048
1)
RT
B-7
1(F
R75
4439
)3
86(4
47/5
15)
uncu
lture
dba
cter
ium
clon
eD
OK
CO
NFY
Mcl
one2
13(D
Q82
8506
)
agri
cultu
rals
oil,
Switz
erla
nd75
(361
/479
)K
ineo
spor
iam
ikun
iens
is(A
B37
7117
)
RT
B-5
2(F
R75
4432
)2
95(4
52/4
73)
uncu
lture
dP
irel
lula
sp.c
lone
CL
5.H
469
(FM
1766
48)
rivu
let,
Har
tzM
ount
ain,
Ger
man
y77
(346
/449
)St
rept
omyc
esth
erm
ovul
gari
s(A
B24
9975
)R
TB
-1(F
R75
4407
)1
85(4
26/4
96)
uncu
lture
dba
cter
ium
clon
e2Y
ML
B03
R(E
F630
323)
Myc
ale
laxi
ssim
am
arin
esp
onge
77(3
66/4
74)
Ferr
ithr
ixth
erm
otol
eran
s(A
Y14
0237
)R
TB
-16
(FR
7544
16)
190
(431
/477
)un
cultu
red
bact
eriu
mcl
one
MD
2896
-B14
(EU
0486
73)
mar
ine
sedi
men
t,So
uth
Slop
e,So
uth
Chi
naSe
a81
(392
/481
)St
rept
omyc
eseu
ropa
eisc
abie
i(A
Y20
7598
)R
TB
-90
(FR
7544
46)
191
(440
/483
)un
cultu
red
bact
eriu
mcl
one
HC
M3M
C90
7CFF
(EU
3739
70)
sedi
men
t,E
ast
Med
iterr
anea
nSe
a78
(349
/447
)T
herm
oleo
phil
umal
bum
(AJ4
5846
2)
(Con
tinu
edon
next
page
)
623
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
TAB
LE
3A
ssig
nmen
tof
clon
ese
quen
ces
ofbi
ofilm
sam
ples
from
the
Rud
as-T
orok
spri
ngca
ve(R
TB
)to
taxo
nom
icgr
oups
and
the
clos
ests
eque
nce
mat
ches
ofkn
own
phyl
ogen
etic
affil
iatio
ns(C
onti
nued
)
Rep
rese
ntat
ive
No.
ofSi
mila
rity
Nea
rest
publ
ishe
dE
nvir
onm
enta
lSi
mila
rity
Nea
rest
desc
ribe
dsp
ecie
scl
one∗
clon
es(%
)∗∗re
lativ
eby
BL
AST
∗so
urce
(%)∗∗
byE
zTax
on∗
Plan
ctom
ycet
esR
TB
-99
(FR
7544
49)
295
(396
/414
)un
cultu
red
bact
eriu
mcl
one
E11
(AB
5186
31)
sedi
men
tof
aw
ater
wel
l,C
hiba
,Jap
an78
(321
/412
)P
lanc
tom
yces
lim
noph
ilus
(AB
UK
0100
0001
)B
acte
roid
etes
RT
B-2
(FR
7544
08)
692
(465
/501
)un
cultu
red
Bac
tero
idet
escl
one
DO
KN
OFE
RT
clon
e636
(DQ
8295
49)
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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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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.
REFERENCESAltschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman
DJ. 1997. Gapped BLAST and PSI-BLAST: A new generation of proteindatabase search programs. Nucl Acids Res 25:3389–3402.
Angert ER, Northup DE, Reysenbach AL, Peek AS, Goebel BM, Pace NR.1998. Molecular phylogentic analysis of a bacterial community in SulphurRiver, Parker Cave, Kentucky. Am Mineral 83:1583–1592.
Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ. 2006. Newscreening software shows that most recent large 16S rRNA gene clone li-braries contain chimeras. Appl Environ Microbiol 72:5734–5741.
Barns SM, Cain EC, Sommerville L, Kuske CR. 2007. Acidobacteria phylumsequences in uranium-contaminated subsurface sediments greatly expand
the known diversity within the phylum. Appl Environ Microbiol 73:3113–3116.
Barton HA, Northup DE. 2007. Geomicrobiology in cave environments: past,current and future perspectives. J Cave Karst Stud 69:163–178.
Barton HA, Taylor NM, Kreate MP, Springer AC, Oehrle SA, Bertog JL. 2007.The impact of host rock geochemistry on bacterial community structure inoligotrophic cave environments. Int J Speleol 36:93–104.
Bennett PC, Engel AS. 2005. Microbial contributions to Karstification. In:Gadd GM, Semple KT, Lappin-Scott HM, editors. Microorganisms and EarthSystems. Advances in Geomicrobiology, Society for General Microbiology(SGM) Symposium, Cambridge University Press 65:345–363.
Canaveras JC, Sanchez-Moral S, Soler V, Saiz-Jimenez C. 2001. Microor-ganisms and microbially induced fabrics in cave walls. Geomicrobiol J18:223–240.
Casanova J, Bodenan F, Negrel P, Azaroual M. 1999. Microbial control on theprecipitation of modern ferrihydrite and carbonate deposits from the Cezallierhydrothermal springs (Massif Central, France). Sediment Geol 126:125–145.
Chelius MK, Moore JC. 2004. Molecular phylogenetic analysis of Archaea andBacteria in Wind Cave, South Dakota. Geomicrobiol J 21:123–134.
Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. 2007. EzTaxon: aweb-based tool for the identification of prokaryotes based on 16S ribosomalRNA gene sequences. Int J Syst Evol Microbiol 57:2259–2261.
Engel AS, Porter ML, Kinkle BK, Kane TC. 2001. Ecological assessmentand geological significance of microbial communities from Cesspool Cave,Virginia. Geomicrobiol J 18:259–274.
Engel AS, Randall KW. 2008. Microbially mediated carbonate dissolution inkarst aquifers, in Sasowsky ID, Feazel CT, Mylroie JE, Palmer AN, PalmerMV, editors. Karst from Recent to Reservoirs, Karst Waters Institute SpecialPublication 14:52–56.
Eross A, Madl-Szonyi J, Csoma A. 2008a. Characteristics of discharge at Roseand Gellert Hills, Budapest, Hungary. Cent Eur Geol 51:267–281.
Eross A, Csoma EA, Madl-Szonyi J. 2008b. The effects of mixed hydrothermaland meteoric fluids on karst reservoir development, Buda Thermal Karst,Hungary. In: Sasowsky ID, Feazel CT, Mylorie JE, Palmer AN, Palmer MV.editors. Leesburg, VA: Karst from Recent to Reservoirs: Special Publication14, Karst Waters Institute. p. 57–63.
Fujisawa A, Tazaki K. 2003. The radioactive microbial mats – In case of Misasahot springs in Tottori Prefecture. In: Kamata N, editor. Proceedings: Interna-tional Symposium of the Kanazawa University 21st Century COE ProgramVol. 1. Kanazawa University, Kanazawa, Japan. p. 328–331.
Groth I, Schumann P, Laiz L, Sanchez-Moral S, Canaveras JC, Saiz-JimenezC. 2001. Geomicrobiological study of the Grotta dei Cervi, Porto Badisco,Italy. Geomicrobiol J 18:241–258.
Hirayama H, Takai K, Inagaki F, Yamato Y, Suzuki M, Nealson KH, HorikoshiK. 2005. Bacterial community shift along a subsurface geothermal waterstream in a Japanese gold mine. Extremophiles 9:169–184.
Holmes AJ, Tujula NA, Holley M, Contos A, James JM, Rogers P, GillingsMR. 2001. Phylogenetic structure of unusual aquatic microbial formations inNullarbor caves, Australia. Environ Microbiol 3:256–264.
Jurado V, Groth G, Gonzalez JM, Laiz L, Saiz-Jimenez C. 2005. Agromycessubbeticus sp. nov., isolated from a cave in southern Spain. Int J Syst EvolMicrobiol 55:1897–1901.
Jurado V, Kroppenstedt RM, Saiz-Jimenez C, Klenk HP, Mouniee D, Laiz L,Couble A, Potter G, Boiron P, Rodrıguez-Nava V. 2009. Hoyosella altamiren-sis gen. nov., sp. nov., a new member of the order Actinomycetales isolatedfrom a cave biofilm. Int J Syst Evol Microbiol 59:3105–3110.
Jurado V, Laiz L, Rodriguez-Nava V, Boiron P, Hermosin B, Sanchez-MoralS, Saiz-Jimenez C. 2010. Pathogenic and opportunistic microorganisms incaves. Int J Speleol 39:15–24.
Konhauser KO. 1998. Diversity of bacterial iron mineralization. Earth Sci Rev43:91–121.
Lane DJ 1991. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M.editors. Nucleic Acid Techniques in Bacterial Systematics. New York: Wiley.p. 115–149.
Dow
nloa
ded
by [
Eot
vos
Lor
and
Uni
vers
ity],
[D
r A
nita
Ero
ss]
at 0
5:56
18
June
201
2
BIOFILM BACTERIAL COMMUNITIES 627
Le Guern C, Baranger P, Crouzet C, Bodenan F, Conil P. 2003. Arsenic trappingby iron oxyhydroxides and carbonates at hydrothermal spring outlets. ApplGeochem 18:1313–1323.
Ludwig W, Bauer SH, Bauer M, Held I, Kirchhof I, Schulze R, SchleiferKH. 1997. Detection and in situ identification of representatives of a widelydistributed new bacterial phylum. FEMS Microbiol Lett 153:181–190.
Macalady JL, Jones DS, Lyon EH. 2007. Extremely acidic, pendulous cavewall biofilms from the Frasassi cave system, Italy. Environ Microbiol9:1402–1414.
Macalady JL, Lyon EH, Koffman B, Albertson LK, Meyer K, Galdenzi S, Mar-iani S. 2006. Dominant microbial populations in limestone-corroding streambiofilms, Frasassi Cave System, Italy. Appl Environ Microbiol 72:5596–5609.
Massol-Deya AA, Odelson DA, Hickey RF, Tiedje JM. 1995. Bacterial commu-nity fingerprinting of amplified 16S and 16–23S ribosomal DNA sequencesand Restriction Endonuclease Analysis (ARDRA). Dordrecht: Kluwer Aca-demic Publishers. p. 3.3.2:1–3.3.2:8.
Messing J. 1983. New M13 vectors for cloning. Meth Enzymol 101:20–78.Miseta R, Palatinszky M, Makk J, Marialigeti K, Borsodi AK. 2012. Phylo-
genetic diversity of bacterial communities associated with sulfurous karsticwell waters of a Hungarian spa. Geomicrobiol J 29:101–113.
Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex microbialpopulations by denaturing gradient gel electrophoresis analysis of polymerasechain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol59:695–700.
Northup DE, Barns SM, Yu LE, Spilde MN, Schelble RT, Dano KE, CrosseyLJ, Connolly CA, Boston PJ, Natvig DO, Dahm CN. 2003. Diverse microbial
communities inhabiting ferromanganese deposits in Lechuguilla and SpiderCaves. Environ Microbiol 5:1071–1086.
Northup DE, Dahm CN, Melim LA, Spilde MN, Crossey LJ, Lavoie KH, Mal-lory LM, Boston PJ, Cunningham KI, Barns SM. 2000. Evidence for geomi-crobiological interactions in Guadalupe caves. J Cave Karst Stud 62:80–90.
Pasic L, Kovce B, Sket B, Herzog-Velikonja B. 2010. Diversity of microbialcommunities colonizing the walls of a Karstic cave in Slovenia. FEMS Mi-crobiol Ecol 71:50–60.
Porter ML, Engel AS, Kane TC, Kinkle BK. 2009. Productivity-diversity rela-tionships from chemolithoautotrophically based sulfidic karst systems. Int JSpeleol 28:27–40.
Portillo MC, Saiz-Jimenez C, Gonzalez JM. 2009. Molecular characterization oftotal and metabolically active bacterial communities of ‘white colonizations’in the Altamira Cave, Spain. Res. Microbiol 160:41–47.
Schabereiter-Gurtner C, Saiz-Jimenez C, Pinar G, Lubitz W, Rolleke S. 2002.Phylogenetic 16S rRNA analysis reveals the presence of complex and partlyunknown bacterial communities in Tito Bustillo cave, Spain, and on its Palae-olithic paintings. Environ Microbiol 4:392–400.
Stackebrandt E, Goebel BM. 1994. Taxonomic note: A place for DNA-DNA re-association and 16S rRNA sequence analysis in the present species definitionin bacteriology. Int J Syst Bacteriol 44:846–849.
Vlasceanu L, Sarbu SM, Engel AS, Kinkle BK. 2000. Acidic cave-wall biofilmslocated in the Frasassi Gorge, Italy. Geomicrobiol J 17:125–140.
Zhou JP, Gu YQ, Zou CS, Mo MH. 2007. Phylogenetic diversity of bacteriain an Earth-cave in Guizhou Province, Southwest of China. J Microbiol45:105–112.
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