Some microbial genera and species found in the artwork of Portinari (Table 1) are common in museums and libraries. Under moderate or humid climate conditions, fungal communities are dominated by Alternaria, Cladosporium, Epicoccus, Aureobasidium and Phoma species. Penicillium, Aspergillus and Fusarium, among others, are also commonly found in books and paintings . The presence of certain species of contaminating fungi can represent, by itself, a threat to human health and to the conservation of cultural heritage, since many of them may present some degree of pathogenicity, in addition to being, mostly, potential cellulolytic agents. In these cases fungi of the genera Cladosporium and Penicilium are included, both found in the present artwork and considered potential pathogenic agents .
However, based on the results obtained and presented in Table 1, the presence unusual fungal species was observed, such as Cladosporium halotolerans, Cladosporium xantocromaticum, Daldinia eschscholtzzi. However, they all contribute to the biodeterioration process. A similar observation was confirmed in a work by Ortiz et al.  who identified wood rot fungal species in eight historic churches in Chile and found several groups of fungi of various genera and species not previously reported in Chile, also demonstrating the great decomposition power of this varied microflora.
The proliferation of fungi in museums is closely determined by the interior climate conditions and the available nutrients, which is directly related to the amount of impurities brought by the air conditioners or the air quality in the external environment. The internal conditions, as indicated by temperature, relative and specific humidities is the most important factor responsible for fungal growth. In spaces where humidity is higher que70% for a period of many weeks or months, a large fungal diversity can be expected being 55% the limit for fungal growth, considered as a standard for countries with cold weather. Thus, climate control should be adjusted to values below this limit, since water availability seems to be adequate for uncontrolled proliferation. Institutions such as museums, galleries, libraries and archives are strongly advised to control temperature and humidity conditions in order to maintain their collections free from mechanical damage and biodeterioration .
Regarding "Índios" official records for the city of Rio de Janeiro indicate that the relative humidity of the air at the date of sample collection was around 64%. However, inside the room there was no control of relative humidity, temperature or light intensity. Such conditions may have worsened the contamination and the disorderly and unpredictable fungal proliferation. As has been reported, even when the relative humidity is maintained around 60%, the works of art or collections of books are not free from contamination and fungal proliferation. The condensation of water occurs on cold surfaces even when they are under low relative humidity, and this represents an opportunity for fungal growth, since they are not very demanding for specific substrates, being able to grow in a wide variety of substrates . The most precious documents of mankind are made of paper, fabrics, papyri and scrolls, as well as artworks of high artistic value. Due to the great diversity of exoenzymes produced by fungi – cellulases, glucanases, lacases, phenolases, keratinases, mono oxygenases, among others – and its remarkable ability to grow in low aw values, the preservation of museum objects is inevitably connected with prevention, monitoring and treatment of the occurrence of fungi in contaminated pieces .
<[>In the present work, the isolation of several species of Penicillium
was made in the three points - air, the artwork itself and edge of the artwork - evidencing the predominance these fungi assume in fungal contamination and proliferation in works of art. Studies show that the genus Penicillium, among most agents that promote biodeterioration, is a ubiquitous organism responsible for modifications of paper support. Being a cellulolytic fungus, it attacks the support of artworks when living in a favorable environment, that is, with favorable temperature and humidity and availability of other essential nutritional elements. Such conditions are very common in libraries and collections that store historical pieces since many of them do not have a rigid control of these conditions, so there may be presence of dirt, humidities from infiltrations and even floods, demolitions . Because they produce acidic pigments, fungi end up generating particular local conditions that modify the physicochemical properties of materials, as seen in the work of Lavin et al.
. In this work, they observed that biofilm formation by Scopularriopsis
sp. and Fusarium
sp., isolated from paper document collections, produced reddish-brown stains, attacked paper structure and produced a pH reduction in the magnitude of one unit, accelerating paper's biodeterioration processes.
The present work also revealed high contamination levels inside the room due to the presence of some species of fungi. The danger of this contamination lies in the fact that spores suspended in the air represent a risk for both human health and constitution of the materials. Its biodeterioration capabilities can potentially cause irreversible damage to artworks and other objects of cultural heritage. It has been reported by other authors that air contamination is the main vehicle for spore’s dissemination. In addition, the co occurrence of the same fungal species in works of art and in air samples is by itself a demonstration of this cross-contamination .
Irradiation of fungal samples at three different doses, 16, 19 and 22 kGy revealed different levels of tolerance between microorganisms. Figures 3 and 4 show the growth on Petri dishes of radiation tolerant species. It can be observed that most of them are dark colored.
Other factors may be involved in the viability of fungi or tolerance to gamma radiation. Multicellular or bicellular spores are more tolerant to gamma radiation than unicellular spores. In addition, the number or density of mycelia in the inoculum exposed to radiation may affect the radiation dose required for the inactivation of the microorganism. Generally, a high density of mycelium in the inoculum requires an elevation of the radiation dose . Shuryak et al.  also studied fungi and yeasts to resist exposure to chronic and acute radiation up to 36 kGy. They concluded that resistance leves were different among organisms from the same order and even species sharing a large core of genes. Basidiomycetes and Ascomycetes constitute a relatively resistant grou with marked differences in acute and chronic radiation doses. Levels of DNA damage production and repair are critical to chronic resistance in replicating cells. On the other hand, DNA damage (particularly double-strand breaks) prevent survival during acute irradiation, as observed in the present work. As previously mentioned, three radiation doses were adopted: 16, 19 and 22 kGy. These values were based on previous studies already conducted and published in the literature that reveal that some species of fungi may be resistant to gamma radiation or may develop some mechanisms of tolerance being therefore difficult to have a complete metabolic inhibition. The results showed that the fungi of the genus Penicillium, Cladosporium, Nigrospora and Curvularia presented a resistance to radiation when the dose of 16 kGy was tested, since, after irradiated, they still presented growth. However, the most resistant of all was the genus Cladosporium, which only presented lack of growth in the dose of 22 kGy (Fig. 5). The results obtained in this study are in agreement with those already presented by other authors who conducted studies with C. cladosporioides. According to Boniek et al. , this was the only species resistant to exposure of gamma radiation and this characteristic may be related to the fact that some strains have the metabolic capacity to produce a dark coloured pigment (the biopolymer melanin) that accumulates within the mycelium and protects against UV rays and ionizing radiation. Other published studies have shown that radiotrophic fungal species use melanin to convert beta and gamma radiation to chemical energy for growth . In fact, according to the results presented in Figures 3 e 4, it was found that the tolerant fungi exhibited a dark color, evidencing the presence of the melanin pigment that may be associated with this resistance mechanism.
The resistance of some fungi common to gamma radiation was also studied by Saleh et al. . Ten species of fungi representing the genera Alternaria, Aspergillus, Cladosporium, Curvularia, Fusarium and Penicillium were examined for their resistance to gamma radiation from a source of 137Cs. In this study, it was found that fungi with melanin hyphae such as Alternaria alternata, Cladosporium cladosporioides, Curvularia lunata and Curvularia geniculata survived after high doses of gamma radiation. The macroconidia of Curvularia and Alternaria sp., which are of a thick wall and contain the pigment melanin, showed resistance and, these two characteristics can probably contribute to the increasing resistance of these species. In another study, it was observed that among all fungi isolated from documents with parchment as support, the most frequent (and more resistant to gamma radiation) genera were Penicillium and Aspergillus . These results corroborate those already presented in this study, where there a high resistance to radiation of the genus Penicillium was observed.
Microbial resistance to gamma radiation was investigated by Múčka et al.  and Neuzilová et al. , using 60Co in order to determine DNA stability and effects on cell membranes. Other authors also studied the effect of 60Co, confirming that the negative effect of the radiation was not linearly dependent on the radiation dose .
It is important to emphasize that there is a concern about the consequences of high radiation doses used in relation to the integrity of the support of artworks. Although no studies have been conducted in the present work, there is a common sense about this matter that only very high doses could affect the integrity of the support. In our work, this was not a concern as the isolated fungi were irradiated on Petri dishes. The literature reports that gamma radiation has already been used in other opportunities to decontaminate paper, showing that doses between 3 and 10 kGy were effective in fungal decontamination, without causing significant damage to the materials. The authors also identified that high doses (around 15 kGy) were tested for disinfection of the paper, without also causing any injury to the support. Even so, there is almost nothing in the literature about the effects of gamma radiation on parchment documents. The authors suggested 5 kGy as a minimum dose to be used for decontamination of parchment documents when the main objective is to decontaminate instead of sterilize . A work carried out by Rizzo et al. and Tomazello [30,31] in the restoration of 17th. Century paintings, considered that the appropriate radiation dose to eliminate microorganisms is 6 kGy, being 25 kGy the standard dose for sterilization . In other studies, it was found that the maximum dose already used on parchment support was 30 kGy since there was no damage to the support. However, the study was not conclusive, as there are many types of supports that need to be tested . Negut et al.  conducted a study on the defects induced by gamma radiation with a 60Co source in historical pigments. The results revealed that the radiation-induced changes in 22 historical pigments. Even after three months after irradiation changes were not significant either because there was in fact no pigment color change or because the changes were reversible. Thus, it was concluded that gamma radiation presented itself as a reliable decontamination treatment. Decontamination techniques using other radiation sources have been studied in search of decontamination techniques that have less harmful effects on the object of cultural heritage to be disinfected.
It was concluded that cosmopolitan fungi, isolated from the air, from a charcoal on paper artwork and fabrics of the artwork can be highly resistant to gamma radiation. The same cosmopolitan fungi that proved to be resistant to radiation are worldwide spread in museums, libraries and archives: Nigrospora, Curvularia, Cladosporium and Penicillium. Even though, the literature reports lower levels of radiation, some species are only eliminated under radiation up to 22 kGy. The mechanism involved in the elimination of fungi is probably associated to DNA damages due to acute irradiation, differently from what is observed under chronic irradiation. The results here observed are useful for similar Portinari’s artworks exposed in museums all over the world, if a decision for a non-destructive decontamination technique is taken.
There is no doubt that the technique used presents a key role in the applicability for artworks with similar characteristics, but it still needs to be studied in collaboration with other control techniques, as some level of damage to the support is expected in some cases. Unfortunately, the artwork studied in the present paper was destroyed in a huge fire in 2018 in the Museu Nacional. However, Brazil still have some artworks by Candido Portinari with the same type of support and technique; this way, results from the present work can be useful to solve biodeterioration processes, probably produced by similar fungi here detected.