Analysis of fungal deterioration phenomena in the first Portuguese King tomb using a multi-analytical approach

Highlights

• Application of a multi analytical approach to study D. Afonso I tomb pathologies.

• High fungal contamination associated with all biodeteriorated areas.

• Black crusts holding lead oxides, anhydrite, syngenite and carbonaceous particles.

• Stone erosions with calcium oxalates.

• Biodeterioration by chromatic changes, stone dissolution and calcium oxalate formation.

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 biodeterioration 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 formation. The establishment of different fungal populations coupled with the deposition of environmental particles contributed to the development of the distinct phenomena detected through differential biodeterioration mechanisms.

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 valuable 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 (CaCO₃) is transformed into gypsum (CaSO₄^.2H₂O) under SO₂ loaded atmospheres. This transformation occurs mainly through the interaction of calcium ions from the carbonate material with the sulphur dioxide atmosphere (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 catastrophic spalling (Ortega-Morales et al., 2019). The accumulation of microorganisms, dust, pollen, pollutants, airborne, soil and carbonaceous 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). Although 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 et al., 2014; Pozo-Antonio et al., 2017; Farkas et al., 2018; Ortega-Morales et al., 2019). A classic example of these distinctive traits can be seen in the study conducted by Aires-Barros et al. (2000) with limestones of the Coimbra region, where the authors characterized and distinguished black crusts in polluted environments due to the presence of metallic elements such as Pb, Ba, Cu, Zn and Ti from black crusts of “fossil” origin rich in Fe and Mn but depleted of Pb, Ti, Zn, Cu and Ba. Furthermore, and in general, “thick” black crusts are considered rich in Ca, Si, and C (Wright, 2002), while “thin” black crusts are characterized by enriched Si and Fe atmospheres (Wright, 2002; Gaylarde and Englert, 2006). On the other hand, the carbonaceous components in these crusts might be a result of the polluted environment (e.g. vehicle exhausts), deposition of additional environmental particles and the presence of microorganisms (thriving in such sites, using the other components as nutrient sources) (Saiz-Jimenez, 1995). From a microbiological perspective, the “thin” black crusts can also harbour diverse cyanobacterial (subtropical and tropical environments) or fungal (moderate environments) populations (e.g. Gaylarde and Englert, 2006; Gaylarde et al., 2007; Ortega-Morales et al., 2019).

Fungi are considered broadly versatile biodeteriogens, being able to thrive on rock inhospitable environments and through their synergistic aesthetic, physical and chemical actions contribute to their biodeterioration (Scheerer et al., 2009; Dakal and Cameotra, 2012; Sterflinger and Piñar, 2013; Gadd, 2017a, b). Fungal biophysical biodeterioration can occur by the contraction and expansion of hyphae, leading to the alterations on the stone integrity (Sterflinger, 2000, 2010; De Leo et al., 2019). On the other hand, chemical biodeterioration occurs as a result of the action of extracellular metabolites, acids and metal-chelating compounds, leading to mineral disruption, dissolution and the possible substitution of the original stone minerals by secondary biomineralization (fungal carbonate stone diagenesis) (Sterflinger, 2000, 2010; Gadd, 2007, 2017a, b). Fungi are also known to interfere with stone aesthetical properties due to their high cell wall melanin contents, their involvement in subaerial biofilm development (Scheerer et al., 2009) and their contribution to inorganic pigments chromatic changes (e.g. Rosado et al., 2014, 2016). Therefore, it is imperative to understand fungal diversity, structural distribution and action mechanisms, in order to correctly diagnose biodeterioration processes and implement safe and efficient control strategies to preserve worldwide monuments (Mihajlovski et al., 2014).

In 2013, UNESCO recognized the “University of Coimbra – Alta and Sofia” (Coimbra, Portugal) as a World Heritage. Many of the monuments at these sites were constructed using limestones from quarries inside the city or from nearby regions such as Ançã and Portunhos (Rola, 2014; Catarino et al., 2019). Unfortunately, some buildings in these areas present several distinctive biodeterioration phenomena still scarcely characterized. During the ongoing limestone biodeterioration studies conducted at this World Heritage site (see Soares et al., 2019a, b; Trovão et al., 2019a, b, c for details and examples), it was noticed the presence of various biodeterioration patterns affecting the limestone relieves adorning the tomb of the first Portuguese King D. Afonso I (UNESCO World Heritage Site and Portuguese National pantheon). According to the ICOMOS Illustrated glossary on stone deterioration patterns (Vergès-Belmin, 2010), these phenomena could be specifically identified as: presence of visual mould proliferation, black crusts and stone erosion and disintegration. Given the extreme importance of this emblematic monument, the aim of this work was to deeply characterize the distinct biodeterioration phenomena on the basis of elemental, mineral, chemical and biological characteristics. For this purpose, a combination of mycological analysis (Illumina MiSeq® and cultivation dependent methodologies) with analytical techniques (colorimetry, microscopy, X-ray fluorescence (XRF), X-ray powder diffraction (p-XRD) and Raman spectroscopy analysis) was applied. This integrative analysis allowed to deeply study the fungal communities structure and diversity, the chemical and mineral tomb composition and the occurrence of potential deterioration products affecting it, providing crucial data that will ultimately allow a well-planned restoration of this ancient and remarkable tomb in the future.