A time travel story: metagenomic analyses decipher the unknown geographical shift and the storage history of possibly smuggled antique marble statues Abstract In this study, three possibly smuggled marble statues of an unknown origin, two human torsi (a female and a male) and a small head, were subjected to molecular analyses. The aim was to reconstruct the history of the storage of each single statue, to infer the possible relationship among them, and to elucidate their geographical shift. A genetic strategy, comprising metagenomic analyses of the 16S ribosomal DNA (rDNA) of prokaryotes, 18S rDNA of eukaryotes, as well as internal transcribed spacer regions of fungi, was performed by using the Ion Torrent sequencing platform. Results suggest a possible common history of storage of the two human torsi; their eukaryotic microbiomes showed similarities comprising many soil-inhabiting organisms, which may indicate storage or burial in land of agricultural soil. For the male torso, it was possible to infer the geographical origin, due to the presence of DNA traces of Taiwania, a tree found only in Asia. The small head displayed differences concerning the eukaryotic community, compared with the other two samples, but showed intriguing similarities with the female torso concerning the bacterial community. Both displayed many halotolerant and halophilic bacteria, which may indicate a longer stay in arid and semi-arid surroundings as well as marine environments. The microbiomes retrieved from each statue showed to be very specific, but some individual members showed to be biological markers for the geographical regions through which the pieces traveled and for the conditions under which they were stored.

Mixed culture: encouraging cross-disciplinary collaboration and communication to enhance learning Abstract Purpose Undergraduate microbiology or biology students are not likely to be acquainted with the deterioration of cultural heritage. However, the topic provides an excellent opportunity for students to bring their existing ‘non-scientific’ skills and knowledge (such as history, geography, art, photography, and textiles) together with applied microbiology in order to develop their learning. Methods A lecture on microbiology and art was given to first-year undergraduate students, who were then set an assignment to investigate a topic of their choice linking the two subjects, illustrated by a creative output. Results The assignment enabled students to demonstrate their creativity, use their different talents, and engage in new learning. It proved very successful as a tool for engaging and inspiring the students to study microbiology and be more explorative in subsequent years. Indeed, for final year studies, some students continued to work on their ‘art project’. Interdisciplinary collaborations and new learning for the author have been additional benefits of this education project, with many varied outputs, including an ‘Atlas of Biodeterioration’, produced by researchers in the field. Conclusion Biodeterioration of cultural heritage provides a rich focus for the development of informative and innovative activities in an educational setting.

Biodeterioration of Roman hypogea: the case study of the Catacombs of SS. Marcellino and Pietro (Rome, Italy) Abstract No information exists on phototrophs growing on the stone surfaces of the Catacombs of SS. Marcellino and Pietro (the site was only recently opened to the public in 2014). Therefore, it was decided to characterise the microbial communities and to compare them with those of the other previously studied catacombs. Moreover, a new non-invasive strategy to reduce the phototrophic growth was tested. Phototrophic microorganisms were investigated under light and confocal laser scanning microscopes from samples collected non-invasively in situ. Tests were carried out to determine the effect of the application of two essential oils (from L. angustifolia and T. vulgaris) on biofilm photosynthetic activity. Laser-induced fluorescence (LIF) and reflectance measurements in the visible range have been used to evaluate respectively, any chemical modification and discolouration on a frescoed stone that may occur after the application of the essential oils. At all the concentrations of essential oils, there was a quasi-immediate, large reduction in photosynthetic activity of the biofilms. At 10% essential oil concentration, there was no detectable photosynthetic activity after 15 min. At 1%, there was a need for two applications and after 5-day activity was undetectable. No effect of the essential oils on the substrate surface properties or colour modification of the fresco has been observed with the LIF prototype. Cyanobacterial typical of Roman catacombs were present in the sites investigated. Innovative and non-destructive strategies, involving the application of a combination of two essential oils, have been successfully tested and developed to prevent biodeterioration of these sites.

Lichens and other lithobionts on the carbonate rock surfaces of the heritage site of the tomb of Lazarus (Palestinian territories): diversity, biodeterioration, and control issues in a semi-arid environment Abstract Purpose Investigations on the lithobiontic colonization of the stone cultural heritage in (semi-)arid regions are needed to address conservation strategies. In this work, lithobiontic communities were examined on the carbonate rock surfaces of the heritage site of the Tomb of Lazarus. We aimed to evaluate their distribution and interaction with the lithic substrate, together with the efficacy of biocidal treatments for their control. Methods Diversity and abundance of lithobionts were surveyed on the Jerusalem stone blocks of three architectural elements. Observations at the lichen-rock interface were carried out by reflected light and scanning electron microscopy. The efficacy against lichens of the widely used biocide benzalkonium chloride (BZC) was compared for different concentrations and application methods, and evaluated by epifluorescence microscopy. Results Chlorolichens were the dominant component of lithobiontic communities, more thoroughly adapted to the semi-arid conditions of the site than mosses and black biofilms of cyanobacteria and dematiaceous fungi. A different structural organization, in terms of thallus thickness and depth of the hyphal penetration component, characterized epilithic and endolithic lichen species, responsible for different deteriogenic activities. Biocidal assays showed that even the methodologies that are usually effective in temperate conditions (as the application of BZC 1.5% by poultice) may not completely devitalize lichens adapted to the stress conditions of semi-arid climates, unless a pervasive biocide diffusion through metabolically active thalli is carefully guaranteed. Conclusion Lithobionts act as biodeteriogens on the semi-arid surfaces of the investigated heritage site. Their removal is thus recommendable, but it needs to be adequately supported with a careful calibration of devitalization strategies.

Ecological and taxonomic characterisation of Trentepohlia umbrina (Kützing) Bornet growing on stone surfaces in Lazio (Italy) Abstract Purpose The colonisation of building material by photosynthetic organisms is highly dependent on the environmental conditions and the nature of the substrate. The growths of red-orange phototrophic biofilms have been widely reported in the literature and have commonly been associated with the order Trentepohliales, whose ecological and taxonomical information needs to be improved. Considering the recurrent presence of such biofilms throughout the Lazio region, we would identify the occurring species and define their favourable environmental conditions, through morphological, genetic and ecological analyses. Results Biofilms were collected across an altitude range of 0 to 860 m asl, occurring from the coast to 60-km inland. A dominant presence of the filamentous terrestrial green alga Trentepohlia umbrina (Kützing) Bornet was confirmed in all sites sometimes mixed with cyanobacteria of the genus Gloeocapsa. The distribution of Trentepohliales is generally given as pan-tropical, but some species, such as T. umbrina, are also distributed in temperate climates. Here, it is reported for the first time a Mediterranean occurrence of the species. Low humidity and light conditions, such as those occurring on vertical surfaces with mainly northern aspects, are the preferred environmental conditions. Coastal areas were more favourable for its growth, probably due to the higher nocturnal condensation that occurs here. Concrete and mortars were particularly bioreceptive to T. umbrina, but marble and trachyte were also colonised under favourable conditions. Conclusions The findings better define the ecological range of this species, suggesting a wider biogeographic distribution, and adding information on morphological features and genetic data.

Characterization of the archaeal and fungal diversity associated with gypsum efflorescences on the walls of the decorated Sorcerer’s prehistoric cave Abstract Purpose This study focuses on analysis of the archaeal and fungal diversity associated with gypsum efflorescences damaging the walls of the Sorcerer’s prehistoric cave registered as a world cultural heritage site. Method Archaeal 16S rDNA and fungal internal transcribed spacer (ITS) clone libraries were constructed and analysed. Results Two thaumarchaeotal OTUs belonging to the Nitrososphaeraceae family dominated the archaeal community (100% of clones). Nitrososphaeraceae are obligate aerobic, chemolithoautotrophic organisms that derive their energy from the oxidation of ammonia and may contribute to primary productivity in the cave. Seven fungal OTUs belonging to Ascomycota and one belonging to Basidiomycota were present. The Cordycipitaceae family, mainly represented by entomophilous fungi, dominated the analysis (66.7% of clones). Conclusion We show that archaeal and fungal OTUs are associated with gypsum efflorescences damaging the walls of the Sorcerer’s cave. The role of these microorganisms in the deterioration of the walls of the cave remains to be determined.

Survey of relevant taxonomic groups for the design of qPCR primers and internal fluorescent probes for whole characterization of subaerial biofilm Abstract Purpose A deep survey of biodeteriogen microorganisms reported on stone monuments in Europe has been performed based on the available literature dating back to over 30 years. Methods The obtained lists for eukaryotic algae, phototrophic and non-phototrophic bacteria, and fungi were sorted by Genera, and corresponding sequences in triplicate were downloaded by nucleotide database Genbank for a number of selected barcoding markers. On the basis of collected bibliometric diversity, multiple nucleotide alignments were produced and primers were designed for a qPCR assay. The aim of the present study was to obtain accurate oligos for the characterization of subaerial biofilms on the basis of the most comprehensive collection of reports and case studies regarding subaerial biofilms, with particular regard to phototrophic and non-phototrophic bacteria, eukaryotic algae, and molds. Result Primers were designed on conserved regions flanking a variable region, specific for each of the studied groups of microorganisms. Standard curve for absolute quantification relative to each group were determined for four markers. Then, variable regions in the alignments were used to design fluorescent internal probes for qPCR aimed for a multiplex reaction in which relative abundance can be determined. Conclusion The authors propose this kind of cost-effective approach in the study of biofilms for the estimation of algae, molds, and bacteria both for direct in situ analysis and in vitro simulation.

Correction to: Novel qPCR probe systems for the characterization of subaerial biofilms on stone monuments The term ‘subaerial biofilm’ (SAB) has been introduced for microbial communities that develop on solid mineral surfaces exposed to the atmosphere.

Phototrophic biofilm communities and adaptation to growth on ancient archaeological surfaces Abstract Purpose Hypogea can be considered under-examined environments as regards microbial biodiversity. New understanding has been gained about the predominant phototrophic microorganisms forming biofilms colonising archaeological surfaces in hypogea. In fact, the description of new taxa has remained elusive until recently, as many biofilm-forming phototrophs possess a cryptic morphology with a lack of specialised cells. Methods A multiphasic study, including cytomorphological and ecological descriptions, genetic and biochemical analysis was carried out on the biofilms colonising hypogean environments around the Maltese islands. Molecular studies were imperative because biodiversity was found to be more complex than that indicated by classical taxonomy. Results The dominant microbial life-form on archaeological surfaces is a compact subaerial biofilm. This study has led to new strains of the eukaryotic microalgal genus Jenufa, and the prokaryotic cyanobacteria Oculatella, Albertania and Nodosilinea being identified as the principal phototrophic biofilm-formers colonising the ancient decorated surfaces of Maltese hypogea. Complex morphologies and elaborate life cycles were eliminated as biodiversity was dictated only by the local contemporary microenvironment. The production of thick multilayered sheaths aided adherence to the substrate, concentrating microbial cells in biofilm formation. Albertania skiophila trichomes were able to glide inside the extracellular matrix. Oculatella subterranea exhibited phototaxis associated with a photosensitive apical cell containing a rhodopsin-like pigment. Conclusion The biofilm provided a protective barrier and an improved chance of survival for cells growing in a low-nutrient, low-light environment. Effective strategies to prevent and control the growth of biofilms on the archaeological surface should take into consideration the adaptation of microorganisms to this particular mode of life.

Seasonal diversity of the bacterial communities associated with petroglyphs sites from the Negev Desert, Israel Abstract Rock art sites in the form of petroglyphs are found in the Negev Desert in southern Israel. These petroglyph sites include thousands of images depicting the life and culture of people in the region over the years. As they are part of the desert environment, these petroglyphs are exposed to natural and anthropogenic weathering processes. In natural rock sites, it is known that climatic conditions influence weathering and biofilm community development (Friedmann in Orig Life 10:223–235, 1980; Li et al. in PloS one 11:(9),163–287, 2016) Therefore, in order to develop tools for preservation and remediation, it is imperative to ascertain possible seasonal diversity of surface-inhabiting bacterial communities. To do so, we undertook to study and characterize the microbial communities inhabiting the rock sites during winter and summer. Next-generation sequencing (NGS) technologies using 16S rRNA gene were implemented on rock-surface slab samples to identify the microbial communities. 16S rRNA gene sequencing data revealed the presence of five main bacterial phyla, among them Actinobacteria, Cyanobacteria, Proteobacteria, Bacteroidetes, and Chloroflexi across the analyzed sites. The predominant phylum was Cyanobacteria (10–60% relative abundance), followed by Actinobacteria (20–60% relative abundance), Proteobacteria (5–35%), Bacteroidetes (0.02–35%), and Chloroflexi (1–12%). There was no significant difference in the bacterial diversity between samples from winter and summer. Five main phyla were observed in samples from the two seasons, winter (December) 2017 and summer (July) 2018. This study provides an important first step in understanding the possible seasonal dynamic of bacterial communities associated with the petroglyphs.