**4. Wall paintings**

Wall painting, as the pictorial technique, encompasses all painting techniques aimed at beautifying wall surfaces. There is no universally accepted definition of fresco, as well as consensus on what techniques can be included in this type of wall painting, however, the term *al fresco* generally refers to paintings made on a fresh lime mortar with mineral pigments mixed with water [44]. On the contrary, in *fresco a secco* or *al secco* technique painting is done on a dry plaster with paints prepared by mixing mineral pigments with various organic binders [32, 45]. Although hallmarked as an extreme type of habitat, painted layer and lime mortar are also considered to be very suitable and bioreceptive substrates for fungal growth. This is due to the mineral composition and porous nature of lime mortar, and the fact that organic and inorganic components of the painted layer represent a suitable niche for the development of a wide range of heterotrophic microorganisms [46].

Fungal infestation of wall paintings can occur from several sources including contaminated indoor air as the main, but also soil and plants of immediate vicinity, visitors, contaminated conservation tools, and indoor hotspots as secondary sources [7]. Whether a certain fungus will be able to colonize the painted layer or mortar depends on the ecological and physiological requirements of a given species. If the requirements are met the process is further controlled by three main factors: nutrient availability, relative humidity, and temperature [32]. The origin of nutrients in fresco painting is related to (1) additives (chaff, wheat paste, barley flakes, animal hair, hemp and flax fibers, egg whites, oils, fats) mixed with mineral and complex fillers of chopped straw and lime mortar; (2) additives used in the preparation of mortar (liquid resins, tar, polymer latex, emulsions, bitumen, milk, olive and linseed oil, lard, animal blood); (3) binders of plant and animal origin mixed with mineral pigments; (4) casein, paraloid mixtures, fixatives and consolidants based on polymer components (cellulose acetates, polyvinyl acetate, polymethyl acrylate, etc.) used in restoration works [32, 45, 47–50]. These organic components determine the richness of the fresco mycobiota. Since the composition of the painted layer and mortar is predominated by inorganic components, its mycobiota differs greatly from the fungal communities established on other painted works of art [45]. Furthermore, heterogeneously pigmented zones of the painted

layer can be considered as selective substrates that condition the development of a specific mycobiota [51]. Using culture-dependent methods, the most commonly documented fungi on painted layer and mortar of wall paintings are Ascomycota of genera *Acremonium*, *Alternaria*, *Arthrinium*, *Aspergillus*, *Aureobasidium*, *Beauveria*, *Botrytis*, *Chaetomium*, *Chrysosporium*, *Cladosporium*, *Curvularia*, *Dreschlera*, *Engyodontium*, *Epicoccum*, *Eurotium*, *Exophiala*, *Fusarium*, *Geomyces*, *Gliomastix*, *Phoma*, *Penicillium*, *Scopulariopsis*, *Sepedonium*, *Sporotrichum*, *Stachybotrys*, *Stemphylium*, *Trichoderma*, *Trichotecium*, *Ulocladium* and *Verticillium* [7, 45, 52–54]. Contamination by fungi from phyla Basidiomycota is rare (e.g. *Coprinus* spp.), while Zygomycota of genera *Mucor* and *Rhizopus* are isolated frequently but are considered only surface contaminants [45, 55].

Species of the genera *Aspergillus*, *Aureobasidium*, *Alternaria*, *Cladosporium* and *Penicillium*, are frequently listed as the most common wall paintings contaminants, as well as the primary fresco deterioration agents in temperate climates [56]. Many *Cladosporium* species are recognized as the main biological agents in the process of biodeterioration of wall paintings since they are able to not only induce brown discolorations, but also penetrate through the entire painted layer all the way to the mortar support [57] (**Figure 1C** and **D**). Many species of the genus *Penicillium* are known to develop and intensely sporulate in a period of only few days to a few weeks on periodically moist fresco paintings [58]. Furthermore, in addition to dominant members of fungal community, species from less represented genera can also significantly contribute to the damage of wall paintings. For example, isolation of *Phoma* species from the surface of painted layer indicates that given wall paintings are in an active process of decay [59]. *Chaetomium*, *Aureobasidium* and *Epicoccum* species, due to strong proteolytic activity, degrade protein binders of the painted layer, which results in the lifting and separation of the painted layer from the support. Likewise, it has been recently contemplated that species of the genera *Mucor* and *Rhizopus* might be more involved in process of biodeterioration of wall paintings than originally considered, as it was shown in in vitro experiments that they are able to degrade protein binders and epoxy resins [60].

Mechanical (1) and chemical (2) activity of fungi directly results in damages to structural and esthetic integrity of fresco paintings. (1) Hyphal penetration, together with formation of fruiting bodies and various modifications of mycelium, increases internal pressure thereby forming new cracks in the painted layer and mortar, as well as expanding the existing ones. Damages caused by mechanical activity are considered by some to be of greater importance compared to changes induced by environmental factors and fungal chemical activity [61]. Furthermore, aside from mechanical activity, damages as the result of change in substrate properties can also incur due to utilizing fresco components as a source of nutrients for fungal growth (2′1) and/or due to secretion and interaction of fungal metabolites with organic and inorganic components of the painted layer and mortar (2′2) [7, 62]. (2′1) Extracellular enzymes break down complex organic components into simpler molecules enabling their absorption and easier penetration of hyphae into the substrate which results in cracking and peeling of the painted layer and mortar. The main enzymes involved in this process are β-glucosidase, phosphatase, lipase, arylsulfatase, esterase, protease and endo-N-acetyl-PD-glucosamidase [63, 64]. (2′2) Excreted organic acids chelate metal ions present in mineral pigments and mortar, resulting in the formation of mineral salts and complex compounds that increase pressure in pores, which leads to cracking, peeling, and loss of fragments of the painted layer and mortar [65, 66]. Additionally, salts stimulate formation of surface irregularities that serve as suitable sites for the settlement of heterotrophic microorganisms, thereby increasing the bioreceptivity of fresco painting [67]. In these circumstances, there is an uncontrolled biofilm development and acceleration of chemical dissimilation activity through

*Fungal Deterioration of Cultural Heritage Objects DOI: http://dx.doi.org/10.5772/intechopen.98620*

oxidation, reduction and transformation of metal ions in pigments, primarily Fe and Mn, but also As, Pb, Cu, Zn and Hg, resulting in the alterations to the original color of the painted layer [53, 68]. Aside from organic acids, very stable and persistent fungal pigments (melanins, mycosporins, quinones, hydroxyanthraquinones and carotenoids) secreted onto the surface induce changes in the original coloration, which is process that depends on the chemical composition of the pigment, environmental conditions and interactions with substrate components [7, 32].
