The phylum Actinobacteria constitutes a significant part of the bacterial population in subterranean environments8,9,10. Previous studies on different Spanish and Italian caves revealed that the special microclimatic conditions together with nutrient availability and the nature of the organic matter are important factors controlling the activity of Actinobacteria in caves9, which have a major role in biogeochemical processes, namely biodeterioration and bioprecipitation of minerals, as recently discussed3,4,5,8,9. It is remarkable that 99% of RNA sequences retrieved from sample SC3 were from Nocardia.

The phylum Planctomycetes and the classes Alphaproteobacteria and Gammaproteobacteria, also with some abundance in the samples, are commonly reported in polluted subterranean environments11.

The genus Nocardia is frequently isolated from soils12 and clinical specimens10. In the last few years, members of Nocardia were isolated from European and Asian caves10. Although the primary reservoir of Nocardia asteroides was thought to be the soil13, N. asteroides was common in the air of all the halls and galleries in Castañar de Ibor Cave, Spain, but absent in the air outside the cave (unpublished data). Literature data show a worldwide distribution of members of the genus Nocardia in subterranean environments.

The genus Pseudonocardia is also common in subterranean environments, such as gold mine caves10. Pseudonocardia halophobica was found in Thai caves14. Pseudonocardia spp. were reported in Malta catacombs15, Carlsbad Cavern, New Mexico, USA16 and in white colonizations in the Spanish Altamira, Ardales and Santimamiñe caves3,8,17. A previous study on these Spanish caves showed that the DGGE patterns of the metabolically active bacterial communities were composed of a major, almost exclusive, band corresponding to Pseudonocardia, in amounts as high as 85.6% and 65.7% for Ardales and Santimamiñe caves, respectively8.

The presence and abundance of Nocardia and Pseudonocardia in subterranean environments may be driven by:

i Organic matter. Cave colonization by Nocardia and Pseudonocardia were related with agricultural and/or livestock activities on the top soil8,9,18. The organic carbon content in most Italian top soils (86.4% of total land area) is ≤ 2%19. Soil organic matter is composed of biodegraded lignin and humic substances, among other macromolecules20,21. Subterranean dripping waters contain these macromolecules as dissolved organic matter (DOM), which can reach the paintings. Nocardia (e.g. Nocardia erythropolis, Nocardia corallina and Nocardia opaca) and Pseudonocardia spp. use humic substances as sole carbon and nitrogen sources22,23,24. In addition, Nocardia has been shown to degrade lignin in soil25. Barton et al.16 found that 80% of the total community of a limestone formation in Carlsbad Cavern was represented by Pseudonocardia. These authors suggested that organic matter in this cave is of a phenolic and aromatic nature, as previously observed in other caves18. ii Phyllosilicates. Phyllosilicates and particularly clay minerals have a high adsorbance potential for macromolecules26. DOM from dripping waters may be adsorbed to clay, hindering the loss of nutrient in an oligotrophic environment and in this way may be accessed by bacteria. Colonization and growth of Pseudonocardia is favoured by the presence of clayey substrata in the Spanish caves of Ardales and Santimamiñe8. In Carlsbad Cavern, to the limestone that supported the Pseudonocardia were associated clay particles16. Etruscan paintings were applied on a thin layer of clay2. The stimulation of clays on the colonization and metabolic activity of Nocardia was well-known time ago27. iii Iron oxides. Hematite was used as red pigment for the paintings2. Iron is required for an optimal growth of Nocardia28,29. Hematite and clays can adsorbe humic substances, thus facilitating the access of microorganisms to carbon sources30.

In conclusion, the abundant actinobacterial colonization of Tomba della Scimmia is promoted by the presence of organic matter and clays on the walls. Iron oxide represents an additional factor favouring growth. This type of colonization was also observed in other Tuscanian tombs with similar constructive features (e.g. Tomba del Colle in the Poggio Renzo Necropolis, Tomba della Pellegrina, near Tomba della Scimmia, etc.). The actinobacteria are involved in the bioprecipitation of minerals4,5,6 (Fig. 3), among other biogeochemical processes, as well as in the biodeterioration of the paintings. In fact, actinobacteria have a broad ability to produce acids from most carbohydrates and cause mineral leaching31.

The identification of active microorganisms7,17,32 can provide clues for an effective control and should be the target in cleaning and restoration processes, when possible. Biocides treatments in caves where used in the past and currently are critically discussed because they seem not to be effective or at least have not long-term action. If we consider the intimate contact between bacteria and mineral layer (Fig. 3), a removal of the dead bacterial biomass, after treatment of the paintings is impossible without damaging the paint layer. This dead biomass will support the growth of saprophyte fungi as secondary invaders as soon as the biocide is degraded or even the biocide used as a nutrient source by some other bacteria32.

Due to the particular Etruscan pictorial technique (application of pigments on a fine clay layer), the only possible action is controlling the microclimate, visits and the input of DOM to the interior of the tomb by acting on top soil grasses and plants (non agricultural use, seasonal harvest of spontaneous grasses, etc.) in order to reduce organic matter decomposition. Similar actions were carried out in Altamira Cave with remarkable results18. Biocide treatments are not recommended in subterranean environments32,33 because they are not effective at short and mid-term, promote further secondary colonizations and increase microbial biodiversity.