coatings Article On the Biodiversity and Biodeteriogenic Activity of Microbial Communities Present in the Hypogenic Environment of the Escoural Cave, Alentejo, Portugal Ana Teresa Caldeira 1,2,3, * , Nick Schiavon 1,2 , Guilhem Mauran 1 ,Cátia Salvador 1 ,Tânia Rosado 1 , José Mirão 1,2,4 and António Candeias 1,2,3   Citation: Caldeira, A.T.; Schiavon, N.; Mauran, G.; Salvador, C.; Rosado, T.; Mirão, J.; Candeias, A. On the Biodiversity and Biodeteriogenic Activity of Microbial Communities Present in the Hypogenic Environment of the Escoural Cave, Alentejo, Portugal. Coatings 2021, 11, 209. https://doi.org/10.3390/ coatings11020209 Academic Editor: Beatriz Prieto Received: 6 January 2021 Accepted: 8 February 2021 Published: 11 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 HERCULES Laboratory, Universidade de Évora, 7000-809 Évora, Portugal; schiavon@uevora.pt (N.S.); guilhem.mauran@mnhn.fr (G.M.); cscs@uevora.pt (C.S.); tania.s.rosado@gmail.com (T.R.); jmirao@uevora.pt (J.M.); candeias@uevora.pt (A.C.) 2 City U Macau Chair in Sustainable Heritage, Universidade de Évora, 7000-809 Évora, Portugal 3 Departamento de Química, Universidade de Évora, Escola de Ciências e Tecnologia, 7000-671 Évora, Portugal 4 Geosciences Department, School of Sciences and Technology, Évora University, Rua Romão Ramalho 59, 7000-671 Évora, Portugal * Correspondence: atc@uevora.pt Abstract: Hypogenic caves represent unique environments for the development of specific microbial communities that need to be studied. Caves with rock art pose an additional challenge due to the fragility of the paintings and engravings and to microbial colonization which may induce chemical, mechanical and aesthetic alterations. Therefore, it is essential to understand the communities that thrive in these environments and to monitor the activity and effects on the host rock in order to better preserve and safeguard these ancestral artforms. This study aims at investigating the Palaeolithic representations found in the Escoural Cave (Alentejo, Portugal) and their decay features. These prehistoric artworks, dating back up to 50,000 B.P., are altered due to environmental conditions and microbial activity inside the cave. Microbial cultivation methods combined with culture-independent techniques, biomarkers’ viability assays and host rock analysis allowed us to better understand the microbial biodiversity and biodeteriogenic activity within the hypogenic environment of this important cave site. This study is part of a long-term monitoring program envisaged to understand the effect of this biocolonisation and to understand the population dynamics that thrive in this hypogean environment. Keywords: cave art; biofilm; microbial diversity; biodiversity; biomarkers 1. Introduction Within the cultural heritage research field, cave rock art plays an essential role in understanding the development of art and, more generally, of human cultural development. Communities inhabiting Western Europe in the Upper Palaeolithic (35,000–10,000 BP) left us numerous examples of cave artworks in the form of paintings, drawings and engravings testifying the development of this unique phenomenon. Many of these artworks have suffered and are suffering chemical, mechanical and aesthetic alterations due to physical, chemical and biological weathering processes induced by the colonization of host rock surfaces by microbial communities [1–3]. In order to preserve cave artworks for future generations, it is therefore essential to develop analytical tools and protocols enabling fast and reliable monitoring of the microbial activity present on decorated walls in prehistoric caves. Previous studies have successfully isolated and identified the presence in several cave environments of pathogenic fungi such as Hitoplasma capsulatum, Microsporum gypseum and Trichophyton mentagrophytes [4], or Trichosporon [5–8]. Beside issues related to a potential threat to human health, fungi and, more generally, micro-organisms are responsible for Coatings 2021, 11, 209. https://doi.org/10.3390/coatings11020209 https://www.mdpi.com/journal/coatings