METHODS Application of Molecular Techniques to the Elucidation of the Microbial Community Structure of Antique Paintings Antonio Santos & Alejandro Cerrada & Silvia García & Margarita San Andrés & Concepción Abrusci & Domingo Marquina Received: 29 April 2009 / Accepted: 29 June 2009 / Published online: 25 July 2009 # Springer Science + Business Media, LLC 2009 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 gradi- ent 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 scan- ning electron microscopy and fluorescent in situ hybrid- isation 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. Introduction Paintings are composed of a wide range of organic and inorganic constituents [11] and provide different ecological niches that may be colonised by a large variety of microorganisms. A number of reviews provide a compre- hensive picture of the role of microorganisms in the degradation of art objects, such as paintings, stone, wood, paper, masonry, leather, parchment, glass and metal [2, 5, 7, 15, 17]. Many of the components of paintings are biodegradable, and those binders and additives (glues, oils, plasticisers, emulsifiers, thickeners, etc.) that facilitate drawing or the application of paint layers are susceptible substrates for microbial colonisation. Furthermore, the support material (paper, wood, wool, silk, etc.) and many components such as polysaccharides, gums, proteins, oils, egg yolk, etc. may easily support microbial growth if favourable environmental conditions (humidity, tempera- ture, pH, light) are met. By contrast, several components of paintings, such us the heavy metals in pigments, have antimicrobial properties and are important agents that inhibit or modulate the growth of microbial populations. In short, environmental conditions combined with rich nutrients in antique pictorial substrates create suitable conditions for the development of bacteria or the reproduc- tion of quiescent fungal spores. As colonisation proceeds, the smooth surface of the painting is degraded and the pictorial layers became rough and susceptible to attack by new microorganisms [7]. Accordingly, the microbial flora is usually the result of successive colonisations by different microorganisms. The alterations suffered by the substrates are the result of modifications of their chemical composi- tion, to which the microorganisms themselves may partially contribute. Microorganism growth on paintings may cause structural damage involving different processes, such us A. Santos : A. Cerrada : C. Abrusci : D. Marquina (*) Department of Microbiology, Biology Faculty, Complutense University of Madrid, José Antonio Novais, 2, 28040 Madrid, Spain e-mail: dommarq@bio.ucm.es S. García : M. San Andrés Department of Painting (Painting and Restoration), Facultad de Bellas Artes, Complutense University of Madrid, 28040 Madrid, Spain Microb Ecol (2009) 58:692–702 DOI 10.1007/s00248-009-9564-2