The aim of this work is the material characterization of wall paintings and biodegradation assessment, including the analysis of microbial growth and the effect of microbial proliferation, in view of their conservation. The methodology was applied to the study of frescoes dated from 1531, located in the ancient parish church of Santo Aleixo, Southern Portugal. The combined use of optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDS) and μ- X-ray diffraction (XRD) showed that the painting palette is composed of red and yellow ochers, malachite, azurite, and bone black. The pigments do not show signs of chemical deterioration, except for malachite, which transformed to black copper oxide (tenorite). The microbiological study allowed the identification of several bacterial strains (e.g., Gram+ cocci, Gram+ bacilli, Actinomycetes sp.), yeast strains, and filamentous fungi of the genera Penicillium, Cladosporium and Aspergillus, among others of the microflora present in the paintings. Their metabolic activity is primarily responsible for the physical disruption of paint layers and underlying mortars. The combined approach using SEM analysis and enzymatic dehydrogenase measurement allowed the evaluation of microflora proliferation and diagnosis of the biodeterioration of the mural paintings. Additionally, the effect of some commercial biocides was evaluated for the predominant strains in order to select the most efficient biocide.

Samples of blue wall paint layers from selected 15th to 18th century religious mural paintings from southern Portugal (Alentejo) have been analyzed using a multi-analytical methodology involving the combination of in situ visible spectro-colorimetry with microanalytical techniques such as optical and scanning electron microscopy and Raman spectroscopy. In situ analyses and micro-sampling were carried out in nine different churches, many in an advanced state of deterioration. The objectives of this study were: (a) to identify and compare the pigments that were used in the blue paint layers across the Alentejo region and through time by analysis of the elemental and mineralogical composition and pictorial techniques, and (b) to correlate the data between the actual color of the paint layer and the state of conservation of the pigments. For the paintings dated from the 16th century forward, the results show a generalized use of smalt blue. To a lesser extent, natural azurite was used despite the geological richness of the region in copper and pyrite ores. In only one painting was an optical blue made of carbon black and lime found. The pigments, pure or mixed with red and yellow ochres, were coarsely ground and used in different concentrations to create three-dimensional effects. These parameters as well as the presence of iron oxides in underlayer paints influence the colorimetric coordinates in the more transparent smalt blue paint layers. The state of conservation of the pigments plays an important role in the alteration of the paint color. A clear example of this is the fading of the smalt blue in several paintings due to lixiviation processes.