Altamira cave Paleolithic paintings harbor partly unknownbacterial communities

Claudia Schabereiter-Gurtner a;�, Cesareo Saiz-Jimenez b, Guadalupe Pin‹ar a,Werner Lubitz a, Sabine Ro«lleke a;1

a Institut fu«r Mikrobiologie und Genetik, Universita«t Wien, Dr. Bohr-Gasse 9, A-1030 Vienna, Austriab Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Seville, Spain

Received 18 February 2002; accepted 20 February 2002

First published online 2 May 2002

Abstract

Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. Thepresent study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain).One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first timemicrobiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCRamplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Theapplied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteriawith potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint,the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of thesequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related toenvironmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% tomembers of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number ofclones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteriain Altamira cave. B 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Altamira cave; Acidobacterium division; Denaturing gradient gel electrophoresis ; 16S rRNA; Paleolithic painting

1. Introduction

Caves are no uniform environments in terms of geolog-ical and geochemical characteristics. Show cave manage-ments including arti¢cial lighting and the maximum num-ber of daily visitors produce striking di¡erences as well.Altamira and Lascaux caves for example, having valuablepaintings, have been visited extensively in the past. Alta-mira cave received up to 3000 visitors per day in the 1970s.However, these visits resulted in severe deterioration andnowadays the caves are closed for the public. Visitors are

not allowed to enter the caves and are shown cave repro-ductions.Caves represent nutrient-poor ecosystems subjected to

stable and low temperatures and although providing ex-treme environments for life, they are inhabited by a varietyof bacteria. Few studies on cultivated microorganisms incaves have been conducted so far, most probably revealingjust a very minor and not representative part of cave pop-ulation. Information available on uncultivated microor-ganisms in caves is scarce [1,2]. Concerning Altamiracave, several cultivation-based studies described the bacte-rial colonization on the rock walls [3,4], the bacterial com-position of the dripping waters [5] and the presence ofactinomycetes [6].The present report is the ¢rst culture-independent study

on the bacterial colonization of one of the valuable Paleo-lithic paintings in Altamira cave. Altamira cave (Santillanadel Mar, Cantabria, Spain) is known as the Sistine Chapel

0378-1097 / 02 / $22.00 B 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 6 8 - 7

* Corresponding author. Tel. : +43 (1) 4277 54675;Fax: +43 (1) 4277 54674.

E-mail address: gurtifox@gem.univie.ac.at (C. Schabereiter-Gurtner).

1 Present address: Genalysis GmbH, Im Biotechnologiepark, TGZ II,D-14943 Luckenwalde, Germany.

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of Quaternary art. The famous Polychromes Hall, locatednear the entrance of the cave and discovered in 1879, isdecorated with a herd of bison surrounded by horses anddeer (about 14 000 years old). For analysis, one sample(Alt9) was directly taken from a Paleolithic painting areain the Polychromes Hall. This was the ¢rst time that mi-crobiologists were permitted to take a little sample materi-al from Altamira paintings. Without prior cultivation, weextracted nucleic acid from the sampled material and an-alyzed the phylogenetic relationship of PCR ampli¢ed andcloned 16S rRNA genes (16S rDNA). Genetic ¢ngerprint-ing of the bacterial community was performed by denatur-ing gradient gel electrophoresis (DGGE), which allows thesequence-speci¢c separation of partial 16S rDNA ampli-cons of the same length in polyacrylamide gels containinga denaturing gradient. 16S rDNA clone libraries werescreened by DGGE and revealed phylogenetic informationon individual bacterial members [7].

2. Materials and methods

2.1. Sampling and DNA extraction

Sample Alt9, a red paint pigment taken from next to apainted bison in the Polychromes Hall in Altamira cave,was taken under the supervision of the cave director byscraping o¡ material with a sterile scalpel. DNA was ex-tracted from less than 1 mg of sample material as de-scribed previously for wall painting material [7]. Cell lysiswas based on lysozyme and proteinase K digestion, SDStreatment and freeze-thawing. Possible PCR inhibitorswere complexed by hexadecyltrimethyl ammonium bro-mide, and genomic DNA was puri¢ed by elution throughsilica gel membranes.

2.2. PCR ampli¢cation of 16S rDNA fragments,construction of 16S rDNA clone libraries and DGGEcommunity ¢ngerprinting

Three microliters of the DNA extract were tested forPCR-ampli¢able DNA with 16S rDNA-speci¢c primers.Fragments corresponding to nucleotide positions 341^926 of the Escherichia coli 16S rRNA gene sequencewere ampli¢ed with the forward primer 341f and the re-verse primer 907r as described previously [7]. 16S rDNAclone libraries were constructed by cloning 5 Wl PCR prod-uct ampli¢ed with primers 341f and 907r. Of the sample,one hundred recombinant clones were randomly screenedfor di¡erent 16S rDNA inserts by DGGE as describedpreviously [7]. Inserts of clones producing PCR productsmatching with the most intense bands and with some faintbands of the DGGE ¢ngerprints were sequenced.For DGGE community ¢ngerprinting, 200-bp frag-

ments corresponding to nucleotide positions 341^534 inthe E. coli 16S rRNA gene sequence were ampli¢ed with

the forward primer 341fGC and the reverse primer 518r[8]. PCR reactions were carried out in 100-Wl volumes with5 Wl PCR product of the ¢rst ampli¢cation as templateDNA. Cycling conditions were as described previously[7]. 200 Wl PCR product, obtained by nested PCR, werepooled, precipitated with 96% ethanol, resuspended in15 Wl sterile and double distilled H2O and separated byDGGE. Gel electrophoresis was performed as describedelsewhere [8] in a linear denaturant gradient from 25 to60% in a D GENE System (Bio-Rad, Munich, Germany).After completion of electrophoresis, the gel was stained inan ethidium bromide solution and documented with aUVP documentation system.

2.3. Sequencing of 16S rDNA inserts and phylogeneticanalysis

Sequencing of clone inserts was performed as describedpreviously [7]. For phylogenetic identi¢cation, the ob-tained sequences were compared with sequences of knownbacteria listed in the EMBL nucleotide sequence database.The FASTA search option for the EMBL database wasused to search for close evolutionary relatives [9]. Acces-sion numbers of clones of Alt9 have been deposited at theEMBL nucleotide database under the accession numbersAJ421890^AJ421911.

3. Results and discussion

Altamira cave is one of the most famous karstic caves inSpain containing valuable Paleolithic paintings. The aimof the present study was to investigate the bacterial com-munity present in a red pigment sample (Alt9) taken fromnext to a painted bison in the Polychromes Hall in Alta-mira cave. Fig. 1 shows the DGGE community ¢ngerprintof sample Alt9. It revealed a complex bacterial communitywith 29 visible DGGE bands. In total, 21 clones weresequenced, and the most closely related sequences in theEMBL nucleotide database were determined. Regardingthe 29 visible DGGE bands present in the community¢ngerprint, the numbers of analyzed clones describedabout 72% of the phylogenetic diversity, although neglect-ing the possible presence of overlapping DGGE bands[10]. Thirty-eight percent of the sequences analyzed werephylogenetically most closely related to cultivated bacte-ria, while the majority (62%) were most closely related toenvironmental 16S rDNA clones, showing the broad spec-trum of unknown and yet to be cultivated bacteria inAltamira cave. Identi¢ed bacteria were related with se-quence similarities between 84.8 and 99.4% to the (i) Pro-teobacteria (52.3%), (ii) green non-sulfur bacteria (4.8%),(iii) Planctomycetales (4.8%), (iv) Cytophaga/Flexibacter/Bacteroides (CFB) phylum (9.5%), (v) Acidobacterium di-vision (23.8%), and (vi) Actinobacteria (4.8%) (Fig. 1).(i) Bacteria a⁄liated with the cosmopolitan Proteobac-

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teria were related to the purple non-sulfur phototrophicRhodobium sp., to the N2-¢xing and chemoorganotrophicAzospirillum sp., to the Aquabacterium sp., to a methyltert-butyl ether-degrading strain PM1 most closely relatedto the Aquabacterium sp., Ralstonia sp., Pseudomonas sp.,Pelobacter sp., Polyangium sp., and furthermore to an un-cultured bacterium from Nullarbor caves (clone wb1N15)most closely related to unclassi¢edMyxococcales, and to asulfur-oxidizing strain OAII2 isolated from a hydrother-mal vent most closely related to the sulfur-oxidizing Thi-oalcalovibrio sp. Of these, Pseudomonas is the only genusthat has already been cultivated several times from Alta-mira cave [3,5,11]. The fact that the identi¢ed bacterialcommunity was dominated by the Proteobacteria is partlyin accordance with previous cultivation-based studies inAltamira cave [3,5]. These studies revealed that Actinobac-teria were the dominant bacteria on the walls and ceilings,whereas the dripping waters were dominated by proteo-bacterial genera such as Acinetobacter, Aeromonas, Chro-mobacterium, Chryseomonas, Enterobacter, Erwinia, Flavi-monas, Flavobacterium, Hafnia, Janthinobacterium,Kingella, Pseudomonas, Serratia, and Xanthomonas [3^6].(ii, iii, iv) Bacteria a⁄liated with the green non-sulfur

bacteria, Planctomycetales and with the CFB-divisionwere most closely related to uncultivated bacteria. Theclosest phylogenetic relatives were an uncultured bacte-

rium from a nitrifying^denitrifying activated sludge (cloneH8), an uncultured bacterium from Australian arid soil(clone 0319-7F4), and an unidenti¢ed bacterium from ac-tivated sludge (clone 1959).(v) The Acidobacterium division was the second domi-

nating identi¢ed phylogenetic group after the Proteobac-teria. Bacteria a⁄liated with this division were agriculturalsoil bacteria (clones SC-I-8 and SC-I-49), an unculturedbacterium from soil (clone RB30), and an uncultured bac-terium from polluted soil (clone WD254). The Acidobac-terium division is a recently described monophyletic phy-lum with the only three so far cultivated speciesHolophaga foetida, Geothrix fermentans and Acidobacte-rium capsulatum [12]. The identi¢cation of members ofthe deep-branching Acidobacterium division in Altamiracave paintings represents one of the ¢rst reports for sub-terranean environments. Similar results on the high abun-dance of this phylogenetic group in karstic caves wereobtained by our culture-independent studies in La Garmacave (accession numbers of 16S rDNA clones areAJ421088, AJ421089, AJ421092, AJ421094-AJ421096,AJ421101, AJ421102, AJ421106, AJ421111, AJ421117,AJ421119, AJ421123-AJ421125, AJ421131) and in TitoBustillo cave [13]. These results con¢rm that members ofthis division are ecologically signi¢cant constituents ofmany karstic caves. Their ecological function and possible

Fig. 1. A: Detail of the ethidium bromide-stained 16S rDNA DGGE community ¢ngerprint of sample Alt9, a red pigment sample taken from next toa bison in the Polychromes Hall, Altamira cave. B: Schematic drawing of the DGGE band patterns of A. Broken lines present DGGE bands whichwere not analyzed. Labeled bands correspond to sequenced and phylogenetically analyzed clones. In parentheses accession numbers of closest phyloge-netic relatives and in square brackets percentage similarities of sequenced clones with 16S rDNA sequences listed in the EMBL database.

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impact on the paintings, from which most of the cloneswere obtained, is unknown at present.(vi) The number of detected Gram-positive bacteria was

signi¢cantly low. The only bacterium a⁄liated with theActinobacteria was an uncultured Crater Lake bacterium(clone CL500-10) most closely related to the Frankia sp.On the one hand, cultivation-based studies revealed thatActinomycetes are represented by a greater part of isolatedbacteria in hypogean environments [6,14], which might onthe other hand be due to selective culture media. So far,cultivated Actinomycetes in Altamira cave belong to thegenera Amycolatopsis, Arthrobacter, Brevibacterium, Kocu-ria, Microbacterium, Micrococcus, Nocardia, Nocardioides,Rhodococcus, and Saccharothrix. Furthermore, our studyrevealed no presence of low G+C Gram-positive bacteria,whereas low G+C Gram-positive genera such as Bacillus,Paenibacillus, and Staphylococcus have already been culti-vated from Altamira cave walls [11].In previous culture-independent studies, DGGE com-

munity ¢ngerprinting and 16S rDNA phylogenetic analy-ses already gave insight into the complex taxonomic diver-sity of bacterial communities associated with medievalwall paintings [15^17]. While in Altamira cave the bacte-rial colonization of the prehistoric painting was dominatedby Proteobacteria and Acidobacteria, the medieval wallpaintings were inhabited by Proteobacteria (Aquaspirillum,Chromohalobacter, Deleya, Erythrobacter, Halomonas,Porphyrobacter, Pseudomonas, Rhizobium, and Salmonel-la), by unidenti¢ed Cytophagales, by Actinobacteria (Ar-throbacter, Actinobispora, Amycolata, Asiosporangium,Frankia, Geodermatophilus, Nocardioides, Promicromono-spora, Pseudonocardia, Rubrobacter, Streptomonospora,Saccharopolyspora, and Sphaerobacter), and by somemembers of Archaea. No members of the Acidobacteriumdivision were detected.The ¢nding of complex and partly unknown bacterial

communities in cave Paleolithic paintings herein reporteddeserves attention, since it has been shown that microor-ganisms can a¡ect painting pigments [18]. Research oncave biodiversity and linking of 16S rRNA informationwith bacterial function on the Paleolithic paintings wouldbe of interest. In general, the degradation and discolora-tion of pigments, as well as the formation of pigmentedbio¢lms, the dissolution of metals by acids and chelatingagents, and biomineralization are some examples of theworst damages triggered by microbial growth [19]. Forinstance, Bacillus spp. and Arthrobacter viscosus, isolatedfrom rock art paintings, were found to reduce hematite(iron oxide), a common pigment, in laboratory cultures[18]. Besides, Geothrix fermentans ^ one out of the threeso far cultivated species of the Acidobacterium division,whose uncultured representatives seem to be so highlyabundant on Paleolithic paintings ^ is a Fe(III)-reducingbacterium [12]. This suggests that bacteria playing a sig-ni¢cant role in both iron oxidation and reduction mightrepresent a potential hazard for the conservation of the

Paleolithic paintings. Although culture-independent-basedstudies allow only vague statements about the physiolog-ical properties and the ecological role of identi¢ed bacte-ria, they allow an almost non-selective re£ection on thebacterial diversity and complexity in particular ecologicalniches such as on Paleolithic paintings, where little ornothing is known about living conditions.

Acknowledgements

This work was founded by the European Commission,Project ENV4-CT95-0104. Dr. J.A. Lasheras, director ofthe Museo Nacional y Centro de Investigacio¤n de Alta-mira, is acknowledged for the facilities provided.

References

[1] Cunningham, K.I., Northup, D.E., Pollastro, R.M., Wright, W.G.and LaRock, E.J. (1995) Bacteria, fungi and biokarst in LechuguillaCave, Carlsbad Caverns National Park, New Mexico. Environ. Geol.25, 2^8.

[2] Engel, A.S., Porter, M.I., Kinkle, B.K. and Kane, T.C. (2001) Eco-logical assessment and geological signi¢cance of microbial commun-ities from Cesspool Cave, Virginia. Geomicrobiol. J. 18, 259^274.

[3] Somavilla, J.F., Khayyat, N. and Arroyo, V. (1978) A comparativestudy of the microorganisms present in the Altamira and La Pasiegacaves. Int. Biodeterior. Bull. 14, 103^109.

[4] Can‹averas, J.C., Hoyos, M., Sanchez-Moral, S., Sanz-Rubio, E.,Bedoya, J., Soler, V., Groth, I., Schumann, P., Laiz, L., Gonzalez,I. and Saiz-Jimenez, C. (1999) Microbial communities associated withhydromagnesite and needle-¢ber aragonite deposits in a karstic cave(Altamira, Northern Spain). Geomicrobiol. J. 16, 9^25.

[5] Laiz, L., Groth, I., Gonzalez, I. and Saiz-Jimenez, C. (1999) Micro-biological study of the dripping waters in Altamira cave (Santillanadel Mar, Spain). J. Microbiol. Methods 36, 129^138.

[6] Groth, I., Vetermann, R., Schuetze, B., Schumann, P. and Saiz-Jime-nez, C. (1999) Actinomycetes in kartic caves of Northern Spain (Al-tamira and Tito Bustillo). J. Microbiol. Methods 36, 115^122.

[7] Schabereiter-Gurtner, C., Pin‹ar, G., Lubitz, W. and Ro«lleke, S.(2001a) An advanced molecular strategy to identify bacterial com-munities on art objects. J. Microbiol. Methods 45, 77^87.

[8] Muyzer, G., De Waal, E.C. and Uitterlinden, A.G. (1993) Pro¢ling ofcomplex microbial populations by denaturing gradient gel electro-phoresis analysis of polymerase chain reaction-ampli¢ed genes codingfor 16S rRNA. Appl. Environ. Microbiol. 59, 695^700.

[9] Pearson, W.R. (1994) Rapid and sensitive sequence comparison withFAST and FASTA. Methods Enzymol. 183, 63^98.

[10] Jackson, C.R., Roden, E.E. and Churchill, P.F. (2000) Denaturinggradient gel electrophoresis can fail to separate 16S rDNA fragmentswith multiple base di¡erences. Mol. Biol. Today 1, 49^51.

[11] Can‹averas, J.C., Sanchez-Moral, S., Soler, V. and Saiz-Jimenez, C.(2001) Microorganisms and microbially induced fabrics in cave walls.Geomicrobiol. J. 18, 223^240.

[12] Ludwig, W., Bauer, S.H., Bauer, M., Held, I., Kirchhof, I., Schulze,R., Huber, I., Spring, S., Hartmann, A. and Schleifer, K.H. (1997)Detection and in situ identi¢cation of representatives of a widelydistributed new bacterial phylum. FEMS Microbiol. Lett. 153, 181^190.

[13] Schabereiter-Gurtner, C., Saiz-Jimenez, C., Pin‹ar, G., Lubitz, W. andRo«lleke, S. (2002) Phylogenetic 16S rRNA analysis reveals the pres-ence of complex and partly unknown bacterial communities in Tito

FEMSLE 10475 23-5-02

C. Schabereiter-Gurtner et al. / FEMS Microbiology Letters 211 (2002) 7^1110

Bustillo cave, Spain, and on its Paleolithic paintings. Environ. Micro-biol. in press.

[14] Groth, I., Schumann, P., Laiz, L., Sanchez-Moral, S., Can‹averas,J.C. and Saiz-Jimenez, C. (2001) Geomicrobiological study of theGrotta dei Cervi, Porto Badisco, Italy. Geomicrobiol. J. 18, 241^258.

[15] Gurtner, C., Heyrman, J., Pin‹ar, G., Lubitz, W., Swings, J. and Ro«l-leke, S. (2000) Comparative analyses of the bacterial diversity on twodi¡erent biodeteriorated wall paintings by DGGE and 16S rDNAsequence analysis. Int. J. Biodeterior. Biodegrad. 46, 229^239.

[16] Pin‹ar, G., Saiz-Jimenez, C., Schabereiter-Gurtner, C., Blanco-Varela,M.T., Lubitz, W. and Ro«lleke, S. (2001) Archaeal communities intwo disparate deteriorated ancient wall paintings: detection, identi¢-

cation and temporal monitoring by denaturing gradient gel electro-phoresis. FEMS Microbiol. Ecol. 37, 45^54.

[17] Schabereiter-Gurtner, C., Pin‹ar, G., Vybiral, D., Lubitz, W. andRo«lleke, S. (2001b) Rubrobacter-related bacteria associated withrosy discolouration of masonry and lime wall paintings. Arch. Micro-biol. 176, 347^354.

[18] Gonzalez, I., Laiz, L., Hermosin, B., Guerrero, B., Incerti, C. andSaiz-Jimenez, C. (1999) Microbial communities of the rock paintingsof Atlanterra shelter (South Spain). J. Microbiol. Methods 36, 123^127.

[19] Ciferri, O. (1999) Microbial degradation of paintings. Appl. Environ.Microbiol. 65, 879^885.

FEMSLE 10475 23-5-02

C. Schabereiter-Gurtner et al. / FEMS Microbiology Letters 211 (2002) 7^11 11