Abstract
This paper uses molecular techniques to describe the microstructure and microbiological communities of sixteenth century artwork and their relationships. The microbiological populations, analysed by denaturing gradient gel electrophoresis (DGGE), were highly influenced by the chemical composition of the pictorial layers detected by energy-dispersive X-ray analysis. DGGE revealed that the diversity of microbial communities was lower in pictorial layers composed of pigments with metals, such as Pb, Cu and Hg, than in those found in pictorial layers without such compounds. The number of cultivable microorganisms, mainly fungi and bacteria, was very low in comparison to those found by DGGE, revealing the presence of both cultivable and as-yet-uncultivated (or not viable) species in the samples analysed. Both fungi and bacteria were present in a non-random spatial distribution. Environmental scanning electron microscopy and fluorescent in situ hybridisation analyses revealed that bacterial populations were usually found in close contact with the surface of the pictorial layers, and fungal populations were located on the bacterial biofilm. This work shows, for the first time, the correlation between the diversity of the microbial populations and the chemical composition of the pictorial layers of an artwork.
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Adriaens A (2005) Non-destructive analysis and testing of museum objects: an overview of 5 years of research. Spectrochim Acta Part B 60:1503–1516
Altenburger P, Kampfer P, Makristathis A, Lubitz W, Busse HJ (1996) Classification of bacteria isolated from a medieval wall painting. J Biotechnol 47:39–52
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
Bitossi G, Giorgi R, Mauro M, Salvadori B, Dei L (2005) Spectroscopic techniques in cultural heritage: a survey. Appl Spectrosc Rev 40:187–228
Bock E, Sand W (1993) The microbiology of masonry biodeterioration. J Appl Bacteriol 74:503–514
Caiola M, Forni C, Albertano P (1987) Characterization of the algal flora growing on ancient Roman frescoes. Phycologia 26:387–390
Ciferri O (1999) Microbial degradation of paintings. Appl Environ Microbiol 65:879–885
Cooksey DA (1993) Copper uptake and resistance in bacteria. Mol Microbiol 7:1–5
Costa R, Gomes NCM, Krögerrecklenfort E, Opelt K, Berg G, Smalla K (2007) Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses. Environ Microbiol 9:2260–2273
Dar SA, Kuenen JG, Muyzer G (2005) Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulphate reducing bacteria in complex microbial communities. Appl Environ Microbiol 71:2325–2330
de la Roja JM, Baonza VG, San Andrés M (2007) Application of Raman microscopy to the characterization of different verdigris variants obtained using recipes from old treatises. Spectrochim Acta A Mol Biomol Spectrosc 68:1120–1125
DeLong EF (2005) Microbial community genomics in the ocean. Nat Rev Microbiol 3:459–469
DeLong EF, Karl DM (2005) Genomic perspectives in microbial oceanography. Nature 437:336–342
Duineveld BM, Kowalchuk GA, Keijzer A, van Elsas JD, van Veen JA (2001) Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. Appl Environ Microbiol 67:172–178
González JM, Sáiz-Jiménez C (2005) Application of molecular nucleic acid-based techniques for the study of microbial communities in monuments and artworks. Int Microbiol 8:189–194
Grayston SJ, Wang SQ, Campbell CD, Edwards AC (1998) Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol Biochem 30:369–378
Griffin PS, Indictor N, Koestler RJ (1991) The biodeterioration of stone: a review of deterioration mechanisms, conservation case histories, and treatment. Int Biodeterior 28:187–207
Head IM, Saunders JR, Pickup RW (1998) Microbial evolution, diversity, and ecology: a decade of ribosomal analysis of uncultivated microorganisms. Microb Ecol 35:1–21
Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y (2006) Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments. J Microbiol Methods 66:165–169
Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180:6774–6793
Huws SA, Edwards JE, Kim EJ, Scollan ND (2007) Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems. J Microbiol Methods 70:565–569
Inoue M, Koyano M (1991) Fungal contamination of oil paintings in Japan. Int Biodeterior 28:23–35
Ionita I (1971) Contributions to the study of the biodeterioration of the works of art and of historic monuments. II. Species of fungi isolated from oil and tempera paintings. Rev Roum Biol Ser Bot 16:377–381
Jeffries P (1986) Growth of Beauvaria alba on mural paintings in Canterbury Cathedral. Int Biodeterior 22:11–13
Karpovich-Tate N, Rebrikova NL (1990) Microbial communities on damaged frescoes and building materials in the Cathedral of the Nativity of the Virgin in the Pafnutii-Borovskii Monastery, Russia. Int Biodeterior 27:281–296
Kowalchuk GA, Gerards S, Woldendorp JW (1997) Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA. Appl Environ Microbiol 63:3858–3865
Kumar PS, Griffen AL, Moeschberger ML, Leys EJ (2005) Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol 43:3944–3955
Machado JC, Tulio GV, Siquiera JF, Rôças IN, Peixoto RS, Rosado AS (2007) On the use of denaturing gradient gel electrophoresis approach for bacterial identification in endodontic infections. Clin Oral Invest 11:127–132
Mantler M, Schreiner M (2000) X-ray fluorescence spectrometry in art and archaeology. X-ray Spectrom 29:3–17
Michaelsen A, Pinzari F, Ripka K, Lubitz W, Piñar G (2006) Application of molecular techniques for identification of fungal communities colonising paper material. Int Biodet Biodegr 58:133–141
Möhlenhoff P, Müller L, Gorbushina AA, Petersen K (2001) Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects. FEMS Microbiol Lett 195:169–173
Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317–322
Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes for 16S rRNA. Appl Environ Microbiol 59:695–700
Myers RM, Maniatis T, Lerman LS (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 155:501–527
Oros-Sichler M, Gomes NC, Neuber G, Smalla K (2006) A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities. J Microbiol Methods 65:63–75
Paternoster G, Rinzivillo R, Nunziata F, Castellucci EM, Lofrumento C, Zoppi A, Felici AC, Fronterotta G, Nicolais C, Piacentini M, Sciuti S, Vendittelli M (2005) Study on the technique of the Roman age mural paintings by micro-XRF with polycapillary conic collimator and micro-Raman analyses. J Cult Herit 6:21–28
Rölleke S, Muyzer G, Wawer C, Wanner G, Lubitz W (1996) Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol 62:2059–2065
Rozak DB, Colwell RR (1978) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365–379
Sandhu GS, Kline BC, Stockman L, Roberts GD (1995) Molecular probes for diagnosis of fungal infections. J Clin Microbiol 33:2913–2919
Schabereiter-Gurtner C, Saiz-Jimenez C, Piñar G, Lubitz W, Rölleke S (2002) Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave, Spain, and on its Palaeolithic paintings. Environ Microbiol 4:392–400
Smalla K, Wieland G, Buchner A, Zock A, Parzy J, Kaiser S, Roskot N, Heuer H, Berg G (2001) Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol 67:4742–4751
Taniguchi A, Hamasaki K (2008) Community structures of actively growing bacteria shift along a north–south transect in the western North Pacific. Environ Microbiol 10:1007–1017
Torsvik V, Salte K, Sorheim R, Goksoyr J (1990) Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl Environ Microbiol 56:776–781
Van der Gucht K, Sabbe K, De Meester L, Vloemans N, Zwart G, Gillis M, Vyverman W (2001) Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes. Environ Microbiol 3:680–690
Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alassatova T (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species specific primers. Appl Environ Microbiol 66:297–303
Ward DM, Weller R, Bateson MM (1990) 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63–65
Acknowledgements
This study was supported by a Banco Santander/Universidad Complutense project PR41/06-14967. We would like to thank the Arzobispado de Toledo for its contribution to this work by providing the studied artwork. We are grateful to Dr. Begoña Torralba and Dr. Consuelo Dalmau from the Complutense University of Madrid and M. Gambino from the EU Erasmus Programme for expert assistance and helpful discussions during this study. Also, we are grateful to Centro de Microscopía Electrónica y Citometría (UCM).
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Santos, A., Cerrada, A., García, S. et al. Application of Molecular Techniques to the Elucidation of the Microbial Community Structure of Antique Paintings. Microb Ecol 58, 692–702 (2009). https://doi.org/10.1007/s00248-009-9564-2
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DOI: https://doi.org/10.1007/s00248-009-9564-2