**5. Canvas oil paintings**

One of the best-known pictorial technique today, oil painting on canvas, emerged in the Middle Ages and has since been one of the most important art expressions, constituting outstanding works of art with important historic and cultural value [69]. Structurally speaking, these works of art are composed of the pictorial layer between the protective covering varnish and the ground (or preparatory layer) spread on a linen canvas. Compared to the other forms of artwork, oil paintings on canvas possibly provide the widest range of microhabitats and nutrients that may be exploited by a large variety of microbial species [70]. Materials that constitute the painting, i.e. the cellulose of the canvas support, organic adhesives (i.e. various animal, fish and plant glues) used in sizing the support, natural varnishes, and the oils used in binding the pigments (linseed, turpentine and other oils) are all composed of organic molecules of high nutritional content that are all easily degraded [32, 63]. These organic molecules encompass sugars, gums, and other polysaccharides, proteins and waxes, but also less chemically defined mixtures of biomolecules, such as egg yolk, bile, and urine, as well [45]. Organic glue pastes used to coat the back of paintings with linen canvas, i.e. "re-lining", may also represent a rich nutrient source [71]. Furthermore, dirt, dust and other environmental contaminants (volatile hydrocarbons released from machinery, respiration and cigarette smoke) deposited on the surface of the oil paintings provide nutrients as well [63].

Given the wide range of organic molecules that are present in oil paintings, many different microorganisms may grow provided inadequate storage and favorable environmental conditions, primarily high relative humidity and temperature, are met [45]. These specific environmental conditions may start and/or accelerate the microbial growth, which otherwise would persist on the obverse and the reverse side of the painting in a dormant metabolic state [70]. Among multitudes of different microorganisms, fungi are notorious for their ability to inhabit and decay paintings due to their enormous metabolic activity and ability to grow at low aw values [32]. However, to the best of our knowledge, despite being one of the most numerous objects exhibited and stored in museums and warehouses worldwide, relatively fewer number of studies to date have been engaged in describing the fungal communities dwelling on canvas oil paintings compared to the other forms of art. Using culture-dependent methods, the most commonly documented fungi on painted surface, canvas and wooden frame are Ascomycota of genera *Alternaria*, *Aspergillus*, *Aureobasidium*, *Botrytis*, *Cladosporium*, *Drechslera*, *Epicoccum*, *Fusarium*, *Penicillium*, *Ulocladium*, *Scopulariopsis*, *Stemphylium*, *Trichoderma* and *Wardomyces*, with occasional observations of teleomorphs from *Chaetomium*, *Emericella* and *Eurotium* genera [32, 63, 69, 70, 72, 73]. Review of the above referenced literature data has highlighted fungi that were most frequently isolated from the infested oil paintings: *Alternaria alternata*, *Aspergillus flavus*, *A. niger*, *A. versicolor*, *Aureobasidium pullulans*, *Chaetomium globosum*, *Cladosporium cladosporioides*, *Eurotium chevalieri*, and *Penicillium chrysogenum*. Aside from the Ascomycota, the only other documented species are Zygomycota of *Cunninghamella*, *Mucor* and

*Rhizopus* genera, but they are in most cases regarded as transients only, i.e. part of the surface dust deposits, and not partaking in the complex process of biodeterioration [32, 74]. Air mycobiota of the depots and exhibition rooms, where paintings are stored and exhibited, respectively, was shown to correlate well with fungal communities observed on the surface of the paintings indicating airborne origin of the infestations. Furthermore, higher density and diversity of the fungal communities on the obverse of the painting is a direct result of the higher amount of airborne fungal propagules deposited on the painted surface due to gravitational settling of propagules from the air [22]. It has to be noted, however, that there are discrepancies between results of the studies utilizing culture-dependent and -independent methods. Results of the limited number of studies on non-culturable fraction of the oil paintings fungal communities showed discrepancies in numbers, with unculturable or not viable part of the community being more dense and prevalent, as well as in composition: Ascomycota of genera *Alternaria*, *Ascoidea*, *Aspergillus*, *Blastomyces*, *Candida*, *Chaetomium*, *Coccidioides*, *Diplodia*, *Eutypa*, *Exophiala*, *Fusarium*, *Gaeumannomyces*, *Histoplasma*, *Marssonina*, *Microsporum*, *Neofusicoccum*, *Paracoccidioides*, *Parastagonospora*, *Penicillium*, *Penicilliopsis*, *Pestalotiopsis*, *Pichia*, *Rasamsonia*, *Lodderomyces*, *Neurospora*, *Sordaria*, *Talaromyces*, *Thermothelomyces*, *Thielavia*, *Trichoderma*, *Tuber*, and *Verticillium* were dominant, followed by Mucoromycota of genera *Phycomyces* and *Lobosporangium* and Basidiomycota of genus *Puccinia* [63, 75, 76].

Fungal induced deterioration of canvas oil paintings can occur on both the obverse and the reverse side. It usually starts on the reverse side as canvas components are more readily degraded than those found on the obverse side. In addition, support polymers and the glue sizing in the canvas act as supplementary substrates for fungal growth [22]. Canvas was shown to be one of the most susceptible painting materials (only surpassed by linseed oil), with the susceptibility depending on the percentage content of cellulose, lignin, and other organic components [74, 77]. The higher the percentage of cellulose and lignin, the more resistant it will be to fungal attack [78]. Due to their ability to produce cellulolytic enzymes responsible for cellulose fibers dissolution, fungi of the genus *Alternaria*, *Aspergillus*, *Cladosporium* and *Ulocladium* are considered to be the main agents of canvas deterioration [63, 69, 76].

On the other hand, the degree of deterioration on the obverse side depends on the oil paints and their mode of application. Varnish, added to provide protection against environmental attacks, is the least susceptible painting material to fungal attack, while many pigments are known to possess antifungal properties [74]. Fungal communities are found to be less dense and diverse in pictorial layers containing pigments with heavy metals (e.g. Pb, Cu and Hg), compared to those found in pictorial layers without such compounds [75]. On the obverse side, hydrolytic activities that the fungi undertake to sustain growth results in the detachment of the paint layer from the support, with further increase in the loss of material happening due to excretion of destructive metabolites, i.e. organic or inorganic acids and the additional production of extracellular enzymes: lipases, esterases, endo-N-acetyl-glucosaminidases and proteases [22]. Such activities lead to formation of structural impairments usually manifested as exfoliation of paint layers, cracking, peeling, formation of paint blisters, detachment of the paint layer from the support, deformations and loss of strength of support. Strongly linked to these damages are the esthetic impairments (preceding the structural damages or forming as a resulting consequence) manifesting as the change of the original coloration due to pigment alterations, biofilm formation on the painted surface or staining as the result of pigment excretion by fungi [75]. Fungi of *Aspergillus* and *Penicillium* genera degrade glues and oil binders, and dissolve paints, contributing

to chromatic alterations of the painted surfaces and detachment of the support, while *Aureobasidium pullulans* is considered by many to be the main biological agent of paint deterioration [1, 45, 69].
