Analytical and Bioanalytical Chemistry

, Volume 406, Issue 3, pp 887–895 | Cite as

Microbial communities analysis assessed by pyrosequencing—a new approach applied to conservation state studies of mural paintings

  • T. Rosado
  • J. Mirão
  • A. Candeias
  • A. T. Caldeira
Research Paper


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.


Biodegradation 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.


  1. 1.
    Garg KL, Jain KK, Mishra AK (1995) Role of fungi in the deterioration of wall paintings. Sci Total Environ 167:255–271CrossRefGoogle Scholar
  2. 2.
    Pangallo D, Chovanová K, Šimonovičová A, Ferianc P (2009) Investigation of microbial community isolated from indoor artworks and air environment: identification, biodegradative abilities, and DNA typing. Can J Microbiol 55(3):277–287CrossRefGoogle Scholar
  3. 3.
    Capodicasa S, Fedi S, Porcelli AM, Zannoni D (2010) The microbial community dwelling on a biodeteriorated 16th century painting. Int Biodeter Biodegr 64(8):727–733CrossRefGoogle Scholar
  4. 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. 5.
    Ciferri O (1999) Microbial degradation of paintings. Appl Environ Microbiol 65(3):879–885Google Scholar
  6. 6.
    Ramírez JL, Santana MA, Galindo-Castro I, Gonzalez A (2005) The role of biotechnology in art preservation. Trends Biotechnol 23(12):584–588CrossRefGoogle Scholar
  7. 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
  8. 8.
    Portillo MC, Gonzalez JM (2009) Comparing bacterial community fingerprints from white colonizations in Altamira Cave (Spain). World J Microbiol Biotechnol 25(8):1347–1352CrossRefGoogle Scholar
  9. 9.
    Schabereiter-Gurtner C, Piñar G, Lubitz W, Rölleke S (2001) Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis. J Microbiol Methods 47:345–354CrossRefGoogle Scholar
  10. 10.
    Justé A, Thomma B, Lievens B (2008) Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food Microbiol 25(6):745–761CrossRefGoogle Scholar
  11. 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
  12. 12.
    Gurtner C, Heyrman J, Piñar G, Lubitz W, Swings J, Rolleke S (2000) Comparative analyses of the bacterial diversity on two different biodeteriorated wall paintings by DGGE and 16S rDNA sequence analysis. Int Biodeter Biodegr 46:229–239CrossRefGoogle Scholar
  13. 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
  14. 14.
    Rantsiou K, Iacumin L, Cantoni C, Comi G, Cocolin L (2005) Ecology and characterization by molecular methods of Staphylococcus species isolated from fresh sausages. Int J Food Microbiol 97(3):277–284CrossRefGoogle Scholar
  15. 15.
    Ercolini D (2004) PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 56(3):297–314CrossRefGoogle Scholar
  16. 16.
    Malik S, Beer M, Megharaj M, Naidu R (2008) The use of molecular techniques to characterize the microbial communities in contaminated soil and water. Environ Int 34(2):265–276CrossRefGoogle Scholar
  17. 17.
    Gonzalez JM, Saiz-Jimenez C (2004) Microbial diversity in biodeteriorated monuments as studied by denaturing gradient gel electrophoresis. J Sep Sci 27(3):174–180CrossRefGoogle Scholar
  18. 18.
    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 195:169–173CrossRefGoogle Scholar
  19. 19.
    Karlsson AO, Holmlund G (2007) Identification of mammal species using species-specific DNA pyrosequencing. Forensic Sci Int 173(1):16–20CrossRefGoogle Scholar
  20. 20.
    Jones RT, Robeson MS, Lauber CL, Hamady M, Knight R, Fierer N (2009) A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISME J 3(4):442–453CrossRefGoogle Scholar
  21. 21.
    Roh SW, Kim K-H, Nam Y-D, Chang H-W, Park E-J, Bae J-W (2009) Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing. ISME J 4(1):1–16CrossRefGoogle Scholar
  22. 22.
    Nam Y-D, Jung M-J, Roh SW, Kim M-S, Bae J-W (2011) Comparative analysis of Korean human gut microbiota by barcoded pyrosequencing. PLoS ONE 6(7):e22109CrossRefGoogle Scholar
  23. 23.
    Ye L, Zhang T (2011) Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing. Environ Sci Technol 45(17):7173–7179CrossRefGoogle Scholar
  24. 24.
    Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363(1–2):83–94CrossRefGoogle Scholar
  25. 25.
    Trama JP, Adelson ME, Mordechai E (2007) Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and pyrosequencing. J Clin Virol 40(4):325–329CrossRefGoogle Scholar
  26. 26.
    Petrosino JF, Highlander S, Luna RA, Gibbs RA, Versalovic J (2009) Metagenomic pyrosequencing and microbial identification. Clin Chem 55(5):856–866CrossRefGoogle Scholar
  27. 27.
    Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11CrossRefGoogle Scholar
  28. 28.
    Leite AMO, Mayo B, Rachid CTCC, Peixoto RS, Silva JT, Paschoalin VMF, Delgado S (2012) Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis. Food Microbiol 31(2):215–221CrossRefGoogle Scholar
  29. 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
  30. 30.
    Cutler NA, Oliver AE, Viles HA, Ahmad S, Whiteley AS (2013) The characterisation of eukaryotic microbial communities on sandstone buildings in Belfast, UK, using TRFLP and 454 pyrosequencing. Int Biodeter Biodegr 82:124–133CrossRefGoogle Scholar
  31. 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
  32. 32.
    Mühling M, Woolven-Allen J, Murrell JC, Joint I (2008) Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities. ISME J 2(4):379–392CrossRefGoogle Scholar
  33. 33.
    Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461CrossRefGoogle Scholar
  34. 34.
    Huang X, Madan A (1999) Cap3: A DNA sequence assembly program. Genome Res 9:868–877CrossRefGoogle Scholar
  35. 35.
    Felsenstein J (1993) PHYLIP (Phylogenetic Inference Package), version 3.5c Department of Genetics. University of Washington, Seattle, USA.Google Scholar
  36. 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
  37. 37.
    Rosado T, Gil M, Mirão J, Candeias A, Caldeira AT (2013) Oxalate biofilm formation in mural paintings due to microorganisms—a comprehensive study. Int Biodeter Biodegr 85:1–7CrossRefGoogle Scholar
  38. 38.
    De Felice B, Pasquale V, Tancredi N, Scherillo S, Guida M (2010) Genetic fingerprint of microorganisms associated with the deterioration of an historical tuff monument in Italy. J Genet 89(2):253–257CrossRefGoogle Scholar
  39. 39.
    Jurado V, Sanchez-Moral S, Saiz-Jimenez C (2008) Entomogenous fungi and the conservation of the cultural heritage: a review. Int Biodeter Biodegr 62(4):325–330CrossRefGoogle Scholar
  40. 40.
    Laiz L, Romanowska-Deskins A, Saiz-Jimenez C (2011) Survival of a bacterial/archaeal consortium on building materials as revealed by molecular methods. Int Biodeter Biodegr 65(7):1100–1103CrossRefGoogle Scholar
  41. 41.
    Pepe O, Sannino L, Palomba S, Anastasio M, Blaiotta G, Villani F, Moschetti G (2010) Heterotrophic microorganisms in deteriorated medieval wall paintings in Southern Italian churches. Microbiol Res 165(1):21–32CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • T. Rosado
    • 1
    • 2
  • J. Mirão
    • 1
    • 3
  • A. Candeias
    • 1
    • 2
  • A. T. Caldeira
    • 1
    • 2
    • 4
  1. 1.HERCULES LaboratoryÉvora UniversityÉvoraPortugal
  2. 2.Évora Chemistry Centre and Chemistry DepartmentÉvora UniversityÉvoraPortugal
  3. 3.Évora Geophysics Centre and Geosciences DepartmentÉvora UniversityÉvoraPortugal
  4. 4.Évora Chemistry Centre and Chemistry DepartmentColégio Luís António VerneyÉvoraPortugal

Personalised recommendations