Short Communication

Received: 14 June 2011 Revised: 22 July 2011 Accepted: 25 July 2011 Published online in Wiley Online Library: 14 October 2011

(wileyonlinelibrary.com) DOI 10.1002/jrs.3053

464

The nature of black stains in Lascaux Cave,France, as revealed by surface-enhancedRaman spectroscopyPedro M. Martin-Sanchez,a Santiago Sanchez-Cortes,b Eduardo Lopez-Tobar,b

Valme Jurado,a Fabiola Bastian,c Claude Alabouvettec and Cesareo Saiz-Jimeneza*

We used surface-enhanced Raman spectroscopy to investigate the chemical composition of the black stains threatening therock-art paintings of Lascaux Cave, Montignac, France. The stains are mainly composed of melanin from the fungus Ochroconissp. and the faecal pellets of the collembolan Folsomia candida. Surface-enhanced Raman spectroscopy is a useful technique forrevealing the structure of unknown macromolecules in cultural heritage research. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: SERS; rock-art paintings; black stains; melanins

* Correspondence to: C. Saiz-Jimenez, Instituto de Recursos Naturales yAgrobiologia de Sevilla, IRNAS-CSIC, Apartado 1052, 41080 Sevilla, Spain.E-mail: saiz@irnase.csic.es

a Instituto de Recursos Naturales y Agrobiologia de Sevilla, IRNAS-CSIC, Apartado1052, 41080 Sevilla, Spain

b Instituto de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006 Madrid,Spain

c UMR INRA-Université de Bourgogne, Microbiologie du Sol et de l’Environment,BP 86510, 21065 Dijon Cedex, France

Introduction

Caves are ecosystems populated by bacteria and fungi.[1–3] Touristvisits increase microbial colonisation and often results in fungaloutbreaks,[4,5] which are of great concern when the caves houserock-art paintings. Lascaux Cave, Montignac, France, suffered onJuly 2001 a fungal outbreak of Fusarium solani.[6] To eliminate theoutbreak, the biocide benzalkonium chloride was used between2001 and 2004. Further biocide treatments were applied in 2008.These treatments appear to somehow determine the appearanceof black stains soon after. In 2007 the black stains on the ceiling,walls, and passage banks were so evident that it became, and stillis, one of the cave’s major problems regarding the conservationof the rock art paintings.The origin and nature of the black stains are unknown. To get

information on the chemical structure of compounds present inthe black stains we used surface-enhanced Raman spectroscopy(SERS), a sensitive technique that results in the enhancement ofRaman scattering by molecules adsorbed on rough metal sur-faces. SERS was necessary to obtain a Raman spectrum of thesesamples to quench their large fluorescence emission, which canbe done thanks to energy transfer process occurring betweenthe adsorbed molecule and plasmonic metal nanoparticles. Theenhancement factor can be as much as 1014–1015, which allowsthe technique to be sensitive enough to detect low concentra-tion of macromolecules. The aim was to characterise the mela-nins or chromophoric macromolecules contributing to the colourof the black stains.[7] Previously, the method was successfullyapplied to the characterisation of natural black macromoleculessuch as soil humic acids.[8] Here we describe the usefulness ofSERS analyses in studies on conservation of cultural heritage.

Experimental

Samples

Samplings of the black stains in Lascaux Cave were carried outbetween 2008 and 2010. The samples were maintained at 5 �C

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until they arrived at the laboratory for processing using differentanalytical techniques. From these samples different strains ofOchroconis sp. were isolated. Strain LXA1 isolated from thePassageway was used for cultivation. For melanin extractionand purification, the fungus was cultured in a liquid malt extractmedium at 22 �C for one week. The protocols used weredescribed elsewhere.[9] Faecal pellets from the collembolanFolsomia candida were collected one month after feeding withOchroconis sp. in the laboratory.

Raman measurements

Surface-enhanced Raman spectroscopy analyses were done by us-ing hydroxylamine Ag nanoparticles prepared by reduction withhydroxylamine.[10] A total of 300 mL of a sodium hydroxide solution(1M) was added to 90mL of a 6�10–2M hydroxylamine hydrochlo-ride solution. Then, 10mL of a 1.11�10–3M silver nitrate aqueous so-lution were added dropwise to the mixture under vigorous stirring.One milligram of the analyzed material was dissolved in 1mL ofNaOH 1M. Then, 5 mL of the extracted sample were added to 485mL of the Ag nanoparticles suspension. The last mixture was acti-vated by addition of 10 mL of 0.5M KNO3, which induced the sub-sequent aggregation. SERS spectra were registered with aRenishaw Raman RM2000, equipped with a charge-coupled device

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Sers Of Black Stains In Lascaux Cave, France

(CCD) camera, using the line at 514 nm of an Ar+ laser as excitationsource. Resulting spectra are the average of 10 scans at 5 s and us-ing a laser power at the sample of 2mW.

Figure 2. (A) SERS spectrum of the melanin from Ochroconis sp., strain

Results and Discussion

To determine the chemical nature of the black stains in LascauxCave, a few samples taken from different halls and galleries(Figs 1(A)–(C)) and the Ochroconis sp. melanin (Fig. 2(A)) were sub-jected to SERS. The spectrum of the melanin of Ochroconis sp.(Fig. 2(A)) shows strong bands at 1610, 1305 and 1249 cm–1, whichare attributed to aromatic C = C, C =O and C–C stretching vibra-tions in polycyclic aromatic compounds and to C–O stretchingvibrations in polyphenols, which can also be seen in lignin[11] orsoil humic substances.[8] These bands may be considered as mar-kers for the presence of the melanin of Ochroconis sp. In the anal-ysis of the black stains taken directly from the cave (Figs 1(A)–(C))these three diagnostic bands were evident at the region of1600–1610, 1300–1310 and 1240–1250 cm�1, suggesting thepresence of fungal melanin. SERS data confirmed the participa-tion of Ochroconis sp. melanin in the formation of the black stains.

The SERS spectrum of this melanin differs from thatobtained by Centeno and Shamir[12] for sepia melanin. Somefungal melanins are usually synthesized by the polymerizationof phenols,[13] while sepia melanin occur by the polymeriza-tion of 5,6-dihydroxyindole.[7] Other fungal melanins investi-gated (Aspergillus niger, Stachybotrys chartarum) exhibiteddifferent SERS spectra (Fig. 3). The spectra of these two mela-nins show bands in the 1615–1510 cm�1 and 1380–1350 cm�1,which can also be seen in the SERS spectra of soil humic

Figure 1. SERS spectra of black stains taken from Lascaux Cave. (A) TheGreat Hall of Bulls, (B) The Painted Gallery, and (C) The Nave.

LXA1. (B) SERS spectrum of a black stain densely populated by collembo-lans taken from the Painted Gallery. (C) SERS spectrum of the faecal pel-lets of F. candida fed in the laboratory with Ochroconis sp.

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substance fractions of low molecular weight.[14] Previous analysesof these melanins by pyrolysis[15] and 13 C NMR[16] demon-strated that A. niger melanin was mainly composed of thepolysaccharide nigeran, a (1! 3), (1! 4)-a-D-glucan. In addi-tion, a few aromatic compounds and pyrolysis products fromproteins and some fatty acids were detected.[15] The melaninof S. chartarum was made up of aliphatic chains that upon py-rolysis yielded alkyl-cyclohexenes, alkyl-benzenes and alkyl-naphthalenes.[15] In general, humic substances and fungalmelanins are made up of mixtures of complex materials, in-cluding polysaccharides, proteins, lipids and aromatic com-pounds, some of whose structural features can be easily rec-ognized using different analytical tools,[15,16] including SERS.

In the SERS spectra of the black stains (Fig. 1), bands attributedto aliphatic chains (1442–1446 cm–1) and to carboxylate (at 1390–1400 cm–1, because of COO– bending and at 930 cm–1, attributedto C–COO– stretching) indicate the presence of fatty acids[17]

from the microorganisms.The bands from fatty acids are not as intense in the SERS spec-

tra compared to the aromatic components of the melaninbecause of the high Raman resonance effect of the aromaticmoieties. In fact, the large Raman resonant effect observedin black stains and melanin leads to the intensification of thebands appearing at 2916 and 2862 cm–1, which correspond tocombination bands of the strong ones appearing below1800 cm–1. In samples where the melanin content is lower (Fig. 1)an increase in the amount of aliphatic fractions and carboxylic

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Figure 3. (A) SERS spectrum of the melanin from A. niger. (B) SERS spec-trum of the melanin from S. chartarum.

Figure 4. Folsomia candida specimens feeding on a black stain locatedin the Apse, Lascaux Cave.

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group is deduced from the intensity of bands at 1446, 1398 and930 cm–1. In addition, the band at 2931 cm–1, assigned to theC–H stretching, is more intense in the latter case because of thehigher amount of the aliphatic fraction. Finally, the appearanceof bands at 1534 and 1159 cm–1 (assigned to C =C and C–Cstretching, respectively) is attributed to carotenoid compoundsin the samples.[18]

The dispersion mechanism of black stains in Lascaux Cave is re-lated to the collembolan F. candida through the body and faeces.This collembolan is a cosmopolitan opportunistic troglophile,largely mycophagous, present in caves all over the world. Abun-dant F. candida specimens were found feeding on the blackstains (Fig. 4). It is remarkable that the SERS bands characterisingthe melanin from Ochroconis sp. (Fig. 2(A)) are the same that areobserved in the black stains abundantly colonized by collembo-lans (Fig. 2(B)). We interpret this coincidence as the consequenceof the grazing of collembolans on Ochroconis sp. mycelia and thedigestion of polysaccharides and proteins in the insect gut, butnot of the melanin. It is released in the faecal pellets, thus produc-ing an enrichment of melanin in the black stain. Maraun et al.[19]

also stated that collembolans find fungal melanins difficult to di-gest, and Böllmann et al.[20] believed that this explains the blackcolour of the faeces. A further demonstration was provided bythe analysis of faecal pellets from F. candida, which exhibitedthe same three characteristic bands found in the Ochroconis sp.melanin SERS spectrum (Fig. 2(C)). Therefore, the appearance ofthe three strong bands in the black stain rich in collembolans,

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with less intense bands in other black stains, reinforces the con-tribution of the Ochroconis sp. melanin to the black colour ofthe stain and the role of the collembolans.

Although collembolans enhance the flow of organic carbonbyfragmentation and comminution (physical restructuring) oforganic matter, the gut of F. candida does not degrade fungalmelanins. It is assumed that the Ochroconis sp. mycelium is selec-tively digested in the collembolan gut, in a manner similar to thatreported for humivorous beetle larva, resulting in the digestion ofpolysaccharide and peptide components but not of the aromaticcomponents.[21]

Melanins are extremely recalcitrant macromolecules andtogether with humic acids tend to accumulate in agriculturalsoils,[7–9,22,23] mostly because of their nonhydrolysable aromaticstructure and high molecular weight. Therefore, the Ochroconissp. melanin and perhaps other dematiaceous fungal melaninsthat are not digested by the collembolans persist longer in thecave walls and sediments as residual materials because of theirresistance to microbial degradation. The evidence in support ofthis hypothesis is the selectively melanin-enriched black stainsbecause of collembolan activity and the presence of the bandscharacteristic of Ochroconis sp. melanin in other black stains.

The high density of collembolans in the cave produces anaccumulation of faecal pellets, where partially digested organicmatter is found as well. The continuous activity of collembolansincrease the dispersal of faecal pellets, thus producing new blackstains on the cave walls in the vicinity of previous stains. There-fore, it appears that the dissemination of black stains throughoutthe cave is also associated with collembolans.

Conclusions

Surface-enhanced Raman spectroscopy is a sensitive techniquethat is particularly useful for cultural heritage studies, wheresampling limitation is imposed by the need to preserve paintingsand other assets. Using SERS, we were able to distinguish therelationship between the black stains that appear on the wallsand ceiling of Lascaux Cave and the melanin of Ochroconis sp.,a fungus isolated from the black stains. In addition, this melaninwas found in the faecal pellets of F. candida, a collembolanfeeding on the black stains. The recalcitrance of melanin tocollembolan digestion resulted in the enrichment and dispersionof faecal pellets through the cave.

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Sers Of Black Stains In Lascaux Cave, France

Acknowledgements

The research was supported by the Ministry of Culture and Com-munication, France, project ’Ecologie microbienne de la grotte deLascaux’. This work was also supported by the Spanish Ministry ofScience and Innovation (Grant FIS2010-15405) and ’Comunidadde Madrid’ through the MICROSERES II network (grant S2009/TIC-1476). This is a TCP CSD2007-00058 paper. The collaborationof the Lascaux restoration team and Alina Moskalik-Detalle forproviding Figure 4 is greatly appreciated.

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