Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod Analysis of fungal deterioration phenomena in the first Portuguese King tomb using a multi-analytical approach João Trovão a,* , Francisco Gil b , Lídia Catarino c , Fabiana Soares a , Igor Tiago a , António Portugal a a Centre for Functional Ecology- Science for People and the Planet, Life Sciences Department, University of Coimbra, Coimbra, Portugal b CFisUC- Center for Physics of the University of Coimbra, Department of Physics, University of Coimbra, Coimbra, Portugal c Geosciences Center, Earth Sciences Department, University of Coimbra, Coimbra, Portugal ARTICLE INFO Keywords: Fungi Limestone Black crusts Indoor environments Biodeterioration ABSTRACT During the ongoing characterization of limestone biodeterioration in the UNESCO World Heritage site of “University of Coimbra – Alta and Sofia” (Coimbra, Portugal), it was brought to our attention that the limestone relieves adorning the tomb of D. Afonso I (First Portuguese King), displayed several signs of distinctive biode- terioration patterns. According to the ICOMOS Illustrated glossary on stone deterioration patterns, these could be characterized as visual mould proliferation, presence of black crusts, stone erosion and disintegration. Due to the invaluable nature of this monument, a multi-disciplinary approach (biological and analytical) to fully characterize these phenomena was applied. The results obtained allowed the characterization of the fungal diversity colonizing this monument, as well as the identification of various deterioration products. The results highlighted that the detected indoor black crusts displayed various characteristics from polluted environments, although lacking the typical association with gypsum. The black crusts were also characterized by the presence of lead oxides, damaging salts and carbonaceous particles. In addition, it could be verified that the erosion phenomena found are a result from fungal mediated calcium carbonate dissolution and calcium oxalate for- mation. The establishment of different fungal populations coupled with the deposition of environmental parti- cles contributed to the development of the distinct phenomena detected through differential biodeterioration mechanisms. 1. Introduction Material decomposition is a key recycling process in nature (Viles, 2012). Nevertheless, when occurring on historical objects, it can lead to inestimable cultural heritage loss through microbiological mediated biodeterioration processes (Gadd, 2017a). When compared to outdoor monuments, climatic conditions in indoor environments are usually more naturally stable, being mainly influenced by human activities, architectural design, ventilation, heating, humidity and general building maintenance practices (Gadd, 2017a). Although these settings can be considered simpler environments, under specific microclimatic conditions, stone structures preserved at these sites can provide valu- able colonization niches for biodeteriorative microbial populations (Dakal and Cameotra, 2012). In fact, poor ventilation, accumulation of moisture and stone heterogeneity can contribute for the accumulation of organic substrates able to support the growth of microbial species fit to survive under these extreme conditions. Black crusts can be considered one of the most common types of deterioration phenomena affecting carbonate stones worldwide. Their development occurs mainly on carbonate stones, where calcite (CaCO 3 ) is transformed into gypsum (CaSO 4 . 2H 2 O) under SO 2 loaded atmo- spheres. This transformation occurs mainly through the interaction of calcium ions from the carbonate material with the sulphur dioxide at- mosphere (e.g. Török and Rozgonyi, 2004; Brimblecombe and Grossi, 2005). The formation of gypsum crusts can further entrap atmospheric particles, promoting the formation of precipitates and leading to cata- strophic spalling (Ortega-Morales et al., 2019). The accumulation of microorganisms, dust, pollen, pollutants, airborne, soil and carbonac- eous particles are the main factors leading to the altered appearance of these singularities (Cultrone et al., 2008; El-Gohary, 2010; Ruffolo et al., 2015; Pozo-Antonio et al., 2017; Gaylarde et al., 2017a,b). Al- though gypsum is considered the main constituent of black crusts, distinct formation processes, composition and morphology have been reported according to the substrate and environment studied (e.g. Rivas https://doi.org/10.1016/j.ibiod.2020.104933 Received 18 November 2019; Received in revised form 18 January 2020; Accepted 11 February 2020 * Corresponding author. CFE - Centre for Functional Ecology – Science for People & the Planet, Department of Life Sciences, University of Coimbra, 3000-456, Coimbra, Portugal. E-mail address: jtrovaosb@gmail.com (J. Trovão). International Biodeterioration & Biodegradation 149 (2020) 104933 0964-8305/ © 2020 Published by Elsevier Ltd. T