Microbial communities analysis assessed by pyrosequencing—a new approach applied to conservation state studies of mural paintings
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The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium, but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.
KeywordsBiodegradation Microbial diversity Culture-dependent methods DGGE Pyrosequencing
The authors wish to thank to Biocant for the pyrosequencing analysis, especially to Dr. Conceição Egas. Tânia Rosado acknowledges Fundação para a Ciência e Tecnologia for financial support (PhD grant, SFRH/BD/65747/2009) through program QREN-POPH-typology 4.1., co-participated by the Social European Fund (FSE) and MCTES National Fund.
- 4.Pepe O, Palomba S, Sannino L, Blaiotta G, Ventorino V, Moschetti G, Villani F (2011) Characterization in the archaeological excavation site of heterotrophic bacteria and fungi of deteriorated wall painting of Herculaneum in Italy. J Environ Biol 32:241–250Google Scholar
- 5.Ciferri O (1999) Microbial degradation of paintings. Appl Environ Microbiol 65(3):879–885Google Scholar
- 7.González JM, Saiz-Jiménez C (2005) Application of molecular nucleic acid-based techniques for the study of microbial communities in monuments and artworks. Int Microbiol 8:189–194Google Scholar
- 11.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(6):2059–2065Google Scholar
- 13.Muyzer G, de Waal EC, Uititerlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59(3):695–700Google Scholar
- 29.Fakruddin M, Chowdhury A, Nur Hossain M, Mannan KSB, Mazumdar RM (2012) Pyrosequencing—principles and applications. Int J Life Sci Pharm Res 2(2):65–76Google Scholar
- 31.Duong LM, Jeewon R, Lumyong S, Hyde KD (2006) DGGE coupled with ribosomal DNA gene phylogenies reveal uncharacterized fungal phylotypes. Fungal Divers 23:121–138Google Scholar
- 35.Felsenstein J (1993) PHYLIP (Phylogenetic Inference Package), version 3.5c Department of Genetics. University of Washington, Seattle, USA.Google Scholar
- 36.Rosado T, Reis A, Candeias A, Mirão J, Vandenabeele P, Caldeira AT (2013) Evora Cathedral: Pink! Why not? In: Ropret EP, Ocepek N (eds) 7th International Conference on the application of Raman spectroscopy in Art and Archaeology, Ljubljana, Slovenia. RAA2013, pp 44–45.Google Scholar