4.2 Stone support and mortars
East wall microphotographs of stones thin-section from the host rock (Fig. 3a,b). The sample consists of a small amount of calcite together with dolomite phase (Fig. 3) and match to a dolomitized limestone. The carbonate matrix contains fine-grained dolomite crystals and recrystallized bioclasts, probably benthic foraminifer’s tests, that appear as small globular grains.
West wall sample (Fig. 3c,d) shows the features of a dolostone. It is composed of a fine to medium crystalline mosaic of dolomite as evidenced by the XRD pattern (Fig. 3e).
This type of dolomite is formed during diagenetic processes when magnesium-rich groundwaters allow the replacement of calcite (CaCO3) with dolomite crystals CaMg(CO3)2 (Fairbridge 1957). Both analysed stone samples correspond to the same lithological unit and the same diagenetic process. The host rock of the cave belongs to a sedimentary succession of shallow marine carbonates of Late Triassic age described at the beginning of the past century as “Dolomia Principale di Castellammare del Golfo”.
East and West 1 Mortars correspond to a matrix of white colour without aggregates distinguishable by naked eye. The thickness of the layer changes on millimetres according to the surfaces of the wall. West 2 Mortar looks constituted by a white matrix with different sizes of aggregates not well rounded and with variable colours, black, grey, and red. The thickness of the layer ranges from 2 cm or less in different areas of the painting.
The microscopic observation of mortars cross sections reveals that East Mortar (EM, Fig. 4a) consists of a fine-grained matrix constituted by 90% of binder fraction and 10% of amorphous and well-rounded aggregates with particles size between 5µm and 500µm with colours varying from white to reddish brown. West 1 Mortar (W1M, Fig. 4b) similar to east mortar consists of a fine-grained matrix constituted by 85% of binder fraction and 15% of amorphous and rounded aggregates between 5µm and 200µm with colours varying from white to reddish brown. West 2 Mortar (W2M, Fig. 4c) consists of a matrix constituted by 60% of binder fraction and 40% of angular and rounded aggregates with particles size between 50 µm and up to 2 mm. The colours change from white and grey to reddish brown. W2M is thus different from the two others.
In order to investigate other possible differences and to individuate influences of the environment, the chemical composition was analysed by IR Spectroscopy and XRD.
The FTIR spectra of the three samples (Fig. 5a) show the characteristic bands of calcite (713, 874, 1396 cm− 1). In addition, the spectra suggest the presence of different compounds based on carbonates (816, 746, 295, 220 cm− 1), sulphates (614, 601, 81 cm− 1) and iron oxides (432, 150 cm− 1). The XRD patterns (Fig. 5b) show that the mineral phases occurring in all the samples correspond to calcite (C), quartz (Q) and hydromagnesite (H). According to the literature, these components are related as follow, calcite to the binder fraction, quartz to the aggregates fraction and gypsum to the alteration products of the mortars (Carozzi 1960). It is noted that in coastal regions, the atmosphere is enriched with particles that are naturally generated by the action of wind on the seawater. These particles contain ionic species, principally chlorides and sulphates. Thus, probably sulphates penetrate into the inner of the mortars through ionic diffusion (Salvadori et al.2003; Rizzo et al. 2008). The knowledge of the presence of gypsum is very important because it is well known that it influences the mechanical stability of the mortars and its presence requires specific approaches of conservation (Grassi et al. 2007; Carretti et al. 2007; García-Vera et al. 2020; Salvadori et al. 2003).
4.3 Pigments and binders
By the observation of the paintings at simple sight, it can be asserted that the application of the pigments was done through thin layers by means of strokes. The use of plane colours and thicker lines are characteristic of the style of the east and west 1 painting.
In the case of the west 2 painting, the style is more developed by the use of veiling and fine lines in order to represent the characters. The pigments were applied directly on a dry plaster, the pigment layer is not homogenous and has thickness less than 70 µm approximately (cross-section is reported in Figure S3 of Support Information).
The investigation of the pigments has been performed by XRF technique, analysing 45 area (2x3 mm each). The map and the list of the analysed points is reported in the Figure S4 of Support Information. All the spectra present similarities of composition among all of them. The calcium (Ca) is the major component in all the investigated area (60–90%). In the case of the bright red, dark red and yellow colours, iron (Fe) is the principal component with a content between 15% and 60%. The arsenic (As) was identified in the red dark pigment with a content up to 1%-2% which could be accountable for the final hue. The major component of green colour is copper (Cu) with 27%. The calcium (Ca) content increases up to 80%-90% in the pink and grey colours, while the iron (Fe) content reduces to 8%; no other representative element is present. Finally, the iron (Fe) content is lower to 5% in the black colour. According to these findings, we can hypothesize that the chromatic palette is based on earth pigments also known as ochre or iron oxides (Helen 2003). As seen directly on the paintings, and as the literature indicates, the final hues were obtained by a mixture of pigments. In the case of the dark red, the presence of arsenic (As) suggests the probable use of Realgar pigment in the mixture. In the case of the pink and grey pigments, the higher content of calcium (Ca) suggests the use of Bianco di San Giovanni or white lime. In the case of the green pigment, it is possible to assert the use of malachite and finally, in the case of the black colour the absence of peculiar XRF signals indicates the use of vegetable carbon as black pigment. The presence of malachite can be associate to the conversion of azurite, which is well note it degrades in presence of humidity (Saunders and Kirby 2004). To summarise, the list of colours, chemical elements, and identified pigments is reported in Table 1.
Table 1
List of colours, chemical elements, and identified pigments.
Colour
|
Chemical elements
|
Pigment
|
Bright Red
|
Fe
|
Red ochre
|
Dark Red
|
Fe, As
|
Red ochre + Realgar
|
Yellow
|
Fe
|
Yellow ochre
|
Pink
|
Ca, Fe
|
Mixture of lime white
|
Grey
|
Ca, Fe
|
Mixture of lime white
|
Green
|
Cu
|
Malachite
|
Black
|
---
|
Carbon black
|
In addition, it is interesting to notice that XRF investigation highlighted a strong difference in the elements present in the two walls. In detail, the chlorine content of the east wall (average value 2.6%) is about three times greater than the west wall (average value 0.8%). This difference can be attributed to the highest exposure of the east wall to the marine aerosol, close to the entrance of the cave, and to the resultant deposition of chlorine salts. As well known the interaction of marine aerosol with building materials causes decay processes (Morillas et al. 2020; Comite et al. 2017; Stefanis et al. 2009; Corvo et al. 2010) and considering the type of the paint it is reasonable to think that it is responsible for the stronger decay and resulting bad conservation of the painting of the east wall.
The organic part of the picture layer (binding and varnish) was investigated by Infrared and NMR Spectroscopy. µFT-IR spectra (Fig. 6) show the presence of both inorganic and organic compounds. The bands at 1396, 874, 713 cm− 1 are ascribable to the calcite, while the bands at 1624, 1105, 596 cm− 1 to gypsum in agreement with the results obtained for the mortars. In addition, the IR spectrum of the yellow colour show the bands at 3524, 3400, 3240 cm− 1 suggesting the presence of the pigment gold ochre (Tortora et al.2016). The IR spectrum of the dark red colour show bands at 2954, 2918, 2851 cm− 1 which could be due to the presence of egg yolk binder (Tortora et al. 2016; Vahur et al. 2016); the bands at 1792, 1733, 1716 cm− 1 could indicate that an oxidation process involving triglycerides of the egg binder took place (Raymond et al. 1993).
The IR spectra of the yellow samples show low intensity bands at the same position previously described.
In order to confirm the presence of egg yolk used as binder, NMR analysis was carried out on the sample collected from the dark red pigment, which had the strongest observable IR bands, to detect cholesterol-related signals that might demonstrate the presence of egg yolk (Spyros and Anglos 2006; Sofia et al. 2014).
The signals of 1H NMR spectrum (Fig. 7a) at 0.76, 0.87, and 1.01 ppm could be due to the methyl groups of cholesterol. The 2D HSQC experiment shows that the 1H 13C correlation peaks positions (Fig. 7b) confirming the presence of cholesterol in the analysed sample. The presence of this molecule, according to Sofia et al 2014, is justified by the fact that the oxidation process involving the fatty acids in the egg binder does not affect the original carbon skeleton of the cholesterol. The presence of the cholesterol methyl groups confirms the use of egg yolk as binding media for the paintings.
Microbial characterization
The microbial survey carried out in the Santa Margherita’s cave was aimed to evaluate the microbial community harboured by this unique environment on one hand, and on the other hand, to give insights about the possible role of microorganisms as a potential risk for paintings conservation. Similarly to what was previously unveiled in related cave environments (Ma et al. 2015; Portillo et al. 2008; Pavlik et al. 2018; Yasir 2018; Alonso et al. 2019), the bacterial community of both east and west walls was represented by Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Gammatimonodota phyla (Fig. 8a).
Firmicutes, mostly featured by Bacillaceae and Planococcaceae members, was the most represented bacterial phylum found in both walls, accounting for 57% (east) and 76% (west) of the entire microbial community (Fig. 8a), likely due to the capability of these bacterial strains of overcoming the challenge exerted by the abundant minerals such as carbonates (CaCO3) and silicates (SiO2) (Laiz et al.2000; Randazzo et al. 2015), which were found in the Santa Margherita’s cave. Indeed, bacilli strains such as Bacillus pasteurii, B. cereus, B. megaterium (all belonging to the Bacillaceae family), are bacterial members deeply investigated as bioconsolidants of limestone (De Muynck et al. 2013), concrete (Kim et al. 2013; Achal et al. 2011), and plaster (Anne et al. 2010), as result of biotic processes that catalyse the hydrolysis of urea and organic acids (i.e., oxalates and acetates) as carbon and energy sources to support urease activities, which will result in the production of carbonates (Dhami et al. 2014; Dick et al. 2006). Since the bacterial surface is negatively charged, microbial cells can acquire calcium atoms from the surrounding environment, which can precipitate with carbonates forming CaCO3 at the cell surface. Here, the egg yolk used as a binder for the dark red pigment might have represented a source of fatty acids to support bacterial survival in an oligotrophic environment and calcite biomineralization, as reported in the case of Bacillus subtilis (Rossi et al. 2006). Moreover, Bacillus strains are also able to elicit oxidation reactions (Lu et al. 2010) to deal with the toxicity derived from iron-containing compounds such as iron oxides (Walujkar et al. 2019) and their hydrated forms (Petrushkova and Lyalikova 1986) used as pigments, therefore reinforcing the presence of these bacterial families as a predominant part of the cave’s microbial community, as well as a potential risk for conservation of wall paintings.
Despite the higher relative percentage of Firmicutes in the west wall than the east one, the latter displayed a more heterogeneous microbial composition, as were also present – with a relative percentage abundance ranging from 1 to 2% (Fig. 8b) – bacterial members belonging to Lachnospiraceae, Paenibacillaceae, Streptococcaceae, Ruminococcaceae, Oscillospiraceae, Acidaminococcaceae, Christanellaceae, and Eubacteriaceae families, which were not found in the microbial community of the west wall. A similar conclusion can be drawn for Bacteroidota, Actinobacteriota, and Gemmatimonodata phyla (Figs. 8a) that, although a minority, were solely present in the east wall. This aspect, alongside the higher percentage abundance of unclassified bacterial species in the east wall (16%) versus the west one (3%), might be ascribed to the far more proximity of the former to the external cave environment, easily allowing microbial colonization to occur in this area. Another contributing factor could derive from the high chlorine extent found in the east area of the cave, explaining its high microbial heterogeneity in terms of bacterial families such as Paenibacillaceae, Streptococcaceae, Ruminococcaceae, Cyclobacteriaceae, Rubrobacteriaceae, Sreptomycetaceae, and Sphingomonadaceae in which are included bacterial strains that can moderately tolerate or even thrive under halophilic conditions (Schabereiter-Gurtner et al. 2001; Remmas et al. 2017; Corral et al. 2020; Chen et al. 2010).
It is also worth noting that some of these bacterial families (i.e., Streptococcaceae, Ruminococcaceae, Moraxellaceae, Streptococcaceae, Oxalobacteriaceae, Eubacteriaceae, Sphingomonadaceae, to name a few) are ascribed to the gut microbiota of wildlife (e.g., bat) (Sun et al. 2020; Gaona et al. 2019) that could occasionally use the cave as a shelter, considering that the site entrance is not protected, thus contributing to the microbiota heterogeneity, as well as constituting a potential risk of cave damaging. Thus, microorganisms that can colonize such an environment are likely to be considered as both indigenous and foreign species, which however possess a versatile metabolism, allowing them to thrive under the most disparate nutritional conditions. In this regard, the Proteobacteria phylum mainly inhabited the west area of the cave, reaching a relative abundance of 13% (Fig. 8a). The Gammaproteobacteria family of Pseudomonadaceae (11%) (Fig. 8b) was the most prevalent, being completely absent in the east zone, which was overall featured by a scarce presence of the Proteobacteria (1%) (Fig. 8). A reasonable explanation for such finding might be due to the emphasized oligotrophy of the west area with respect to the east one, thus allowing Gammaproteobacteria to survive exploiting ions present in the rock likely for chemolithotrophic energy production (Schabereiter-Gurtner et al. 2002). Moreover, bacterial members belonging to the Pseudomonadaceae family are peculiarly proficient in forming biofilm on a vast array of surfaces (Harrison et al. 2004), therefore gaining resistance traits to diverse stressors, as those that might derive by the presence of iron oxides, carbonates, and silicates. For instance, Pseudomonas aeruginosa has been described as capable of colonizing different types of silicates growing as a biofilm on mineral surfaces (Aouad et al. 2008), concomitantly stimulating iron release, and overcoming iron deficiency by producing iron scavenging molecules known as siderophores (Hersman et al. 2011). Besides, Pseudomonas strains have been described for their ability to precipitate calcite through biomineralization processes mediated by extracellular urease activities (Abdel-Aleem et al. 2019).