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Black patinas are very common biological deterioration phenomena on lapideous artworks in outdoor environments. These substrates, exposed to sunlight, and atmospheric and environmental agents (i.e. wind and temperature changes), represent extreme environments that can only be colonized by highly versatile and adaptable microorganisms. Black patinas comprise a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms hinders their identification by optical microscopy. This study used Next-Generation Sequencing (NGS) (including shotgun and amplicon sequencing) to characterize the black patina of the travertine embankments (muraglioni) of the Tiber River in Rome (Italy). Overall, the sequencing highlighted the rich diversity of bacterial and fungal communities and allowed the identification of more than one hundred taxa. NGS confirmed the relevance of coccoid and filamentous cyanobacteria observed by optical microscopy and revealed an informative landscape of the fungal community underlining the presence of microcolonial fungi and phylloplane yeasts. For the first time high-throughput sequencing allowed the exploration of the expansive diversity of bacteria in black patina, which has so far been overlooked in routine analyses. Furthermore, the identification of euendolithic microorganisms and weathering agents underlines the biodegradative role of black patina, which has often been underestimated. Therefore, the use of NGS to characterize black patinas could be useful in choosing appropriate conservation treatments and in the monitoring of stone colonization after the restoration interventions.

The preservation status of an underwater cultural site can be determined as the combination of two primary factors, namely the site physical integrity, which results from the past and present interaction of the site itself with the biological/chemical agents located in the surrounding environment, and the exposure of the site to human-related threats. Methods to survey underwater archaeological sites have evolved considerably in the last years in order to face the challenges and problems in archaeological prospection, documentation, monitoring, and data collection.This paper presents a case-study of an archaeological documentation campaign addressed to study and monitor the preservation status of an underwater archaeological site by combining the quantitative measurements coming from optical and acoustic surveys with the study of biological colonization and bioerosion phenomena affecting ancient artefacts. In particular, we present the first results obtained in the survey and documentation campaign carried out during the spring – summer 2018 in the “Nymphaeum of Punta Epitaffio” located in the Marine Protected Area - Underwater Park of Baiae (Naples).

Boring sponges are among the most important erosive organisms causing relevant damage on calcium carbonate substrates in marine environments. The present work offers a contribution to the knowledge of the bioerosive impact of sponges on archaeological artifacts such as the marble statues recovered from the shipwreck of Antikythera (Greece) and the Blue Grotto (Capri, Italy). These peculiar substrates lie on the seabed, but only little information is available about the risks during their underwater life. Bioeroding sponges caused the loss of substantial parts of the artifacts’ lithic substrate (penetrating up to some centimeters depth), often in association with microborers and boring bivalves. The results highlighted that the bioeroding species in Capri were mostly Dotona pulchella mediterranea, Cliona janitrix,Cliona schmidtii, and Spiroxya levispira. Their chambers were often filled by secondary, non-eroding sponges such as Agelas oroides,Jaspis incrustans, Dercitus (Stoeba) plicatus, Erylus sp., and Pachastrella monilifera. In the Antikythera statues, C. schmidtii was the most frequent species, often easily recognized by its purple color. It was often associated with other excavating sponges such as Cliona vermifera, and Dotona pulchella mediterranea. Pachastrella monilifera and Jaspis incrustans were also present as insinuating species. Incomplete sets of spicules and bioerosion pits observed by SEM were referred to the generaAlectona and Siphonodictyon. The presence of the sciophilous epilithic speciesPetrobiona massiliana and Merlia normani in both sites revealed that these archeological sites have comparable environmental conditions. The study allowed the identification of the sponges involved in the colonization of calcareous artifacts and the definition of the important role they played in the damage of the statues.

The shipwreck of Bajo de la Campana (VII–VI century B.C.) was a Phoenician merchant ship accidentally discovered in the 1950s off the coasts of the Murcia region (Spain). Sixty-four elephant tusks were part of the cargo. Some of them were recovered by archaeologists between 2007 and 2011 and are now stored in the restoration laboratory of the National Museum of Underwater Archaeology (ARQVA) of Cartagena. This study investigated the bioerosion traces present on 12 selected tusks in order to hypothesize which marine or terrestrial macroborers could have attacked this substrate. No work has previously looked at the biological degradation of this material. Taking into account the mineral composition of ivory, the hypothesized bioeroders were selected from those reported in the literature as bioeroders of rocks or other hard substrates (bones, corals, shells, etc.). The hypothesized biodeteriogens belongs to several groups of marine invertebrates (echinoids, barnacles, molluscs, sponges, polychaetes, and bryozoans) and terrestrial insects. Unfortunately, the absence of parts of the bioeroders’ body or of skeletal elements inside the studied traces did not allow definitive identification, so the attributions remain hypotheses. However, this study could be considered a starting point for an interesting debate and for future investigations on the bioerosion of this precious material.

This article takes into account the biological colonisation of the marble statues of the famous shipwreck of Antikythera, a merchant ship dated to the first century BC and discovered in the 1900 off Point Glyphadia of the Greek Island of Antikythera. In particular, the study of the epilithic bioencrustations and the environmental interpretation of the benthic community were carried out to understand the underwater life of the statues. Bryozoans, serpulids, brachiopods, calcareous demosponges and red algae were the most abundant components of the community. Most of the identified species were sciaphilic and/or indicative of coralligenous and cave habitats, well suited to the dim light environment of the site (depth of 50–64 m, arrangement of the statues in piles or inside crevices or under boulders). The obtained data were useful to hypothesize the relationship of the artefacts with the marine environment. In particular, the state of preservation of the bioencrustations found on the statues and the presence of superimpositions indicated that colonisation episodes had happened in different time lapses, possibly very distant from each other, but within the same benthic environment.

The study investigated the role of boring polychaetes in the bioerosion of a submerged Roman mosaic floor in the Underwater Archaeological Park of Baiae (Naples, Italy). Three boring species, Dodecaceria concharum, Polydora ciliata, and Pseudopolydora antennata, were found. The initial colonization phases of boring polychaetes were investigated on experimental limestone panels placed underwater in the same marine area. The results showed that the ecological succession was characterized by a first stage of colonization with abundant spionids and a second stage with a boring mature community dominated by D. concharum. The study of silicone casts of the bored traces allowed confirmation that the ichnospecies belonging to the ichnogeneraMaeandropolydora could be attributed to the action of spionid worms, whereas D. concharum is able to produce tongue- and ribbon-shaped borings (ichnogenus Caulostrepsis), and variously contorted galleries (ichnogenus Maeandropolydora) by settling inside borings produced by other polychaetes and increasing the complexity of the gallery system by modifying them. The study of the epilithic polychaete community highlighted that the site is characterized by a low hydrodynamism. Therefore, the most suitable in situ preservation interventions would be the covering of the mosaics with sand layers or geotextiles.

The present study analyzed the dark patina present on the surface of a marble statue exposed in the Gardens of the Quirinal Palace in Rome, with a special focus on euendolithic black meristematic fungi. The study of the spatial distribution of microorganisms and their identification were performed by using a multistep approach based on microscopy, cultural analyses, molecular techniques, and embedding resin-casting technique. Black meristematic fungi were observed in the patina. Since morphological features are not sufficient to identify fungi belonging to this group, cultural and molecular analyses were performed. The results highlighted the presence of the species Coniosporium apollinis and of strains related to the genus Knufia (order of Chaetothyriales). The resin-casting embedding technique demonstrated the active penetration of these fungi into the marble grains, thus documenting their true endolithic behavior. The involvement of phototrophic microorganisms present on the surface in the active penetration of the marble was excluded.

The endolithic activity of the green alga Acetabularia acetabulum (Linnaeus) P.C. Silva, 1952 (Ulvophyceae, Dasycladales, Polyphysaceae) was documented on different lithotypes recovered in the Underwater Archaeological Park of Baiae (Naples). The results show that rhizoids of A. acetabulum penetrate several micrometres deep into marble and limestone creating characteristic ramified boreholes, while they colonized only the surface of brick. Resin casts of tunnels bored by rhizoids, known as the ichnospecies Fascichnus grandis Radtke, 1991, were described in detail. The study reports for the first time the significant role of A. acetabulum in the bioerosion process of underwater archaeological artefacts.

The Pyramid of Caius Cestius in Rome is a funerary architecture erected between 272 and 279 AD. The Pyramid is composed by a nucleus in opus caementicium covered by Luni marble slabs. The marble surface has been colonized by a biocoenosis of microorganisms. The most frequent alteration found on the surface of the stone is the grey-black microbial crust, consisting of three different systematic groups: coccoid and filamentous Cyanobacteria, belonging mainly to the genera Chroococccus , Gloeocapsa and Tolypothrix , and green algae often associated with microscopic fungi or lichenized. The present work is aimed to establish which taxa present in the black crust are epilithic and which ones are endolithic. The cyanobacterium Chroococcus lithophilus is the most abundant and frequent taxon in all the samples collected; its growth is epilithic, chasmoendolithic, cryptoendolithic and euendolithic. The resin embedding-casting technique allowed to define the traces produced by this cyanobacterium highlighting its ability to actively dig tunnels into the marble. The present paper is a significant contribution to the knowledge of the important role of C. lithophilus in the biodeterioration of stone monuments.

This research involves the study of the bioerosion on mosaic fragments recovered from the Underwater Archaeological Park of Baiae (Naples, Italy) caused by the boring sipunculans (Protostomia) and represents the first report of damage by Aspidosiphon muelleri Diesing 1851 on submerged Cultural Heritage. The present study defines the role of this organism in the deterioration of archaeological artefacts. The characteristic traces produced by the action of these organisms on stone materials are described. A set of criteria was established to distinguish the traces of these organisms from those produced by Polychaetes and Clionaid sponges.

Submerged stone archaeological artefacts are bioeroded by endolithic microbiota (cyanobacteria, algae and fungi) and macroborers (Porifera, Bivalvia and Sipuncula). Optical microscope and SEM observations permit to analyse the bioerosion traces and to identify bioeroders. Data obtained with these techniques cannot be used to estimate volumes of material bioeroded. This aspect require the need to collect three-dimensional, close-range data from artefact. In this work we illustrate two 3D imaging techniques used to study bioerosion phenomena of underwater Cultural Heritage. In particular Digital Video Microscope permit the elaboration of 3D images, which are widely employed for close-range acquisitions. Underwater Laser Scanner documents the in situ degradation of submerged artefacts. This research aims to sensitize specialist figures in the study 3D offering a starting point for future collaborations that could lead to interesting results.

This research has been conducted on archaeological artifacts collected from the Grotta Azzurra (Capri, Naples, Italy). Endolithic microbiota and boring Porifera responsible for bioerosion and micro-erosion were analysed. SEM observations permitted the analysis of biodegradation. Embedding casting procedure with polyester resins allowed the identification of three different microborer traces, corresponding to green algae, fungi and boring sponges. According to spicule complement two excavating species and other four sponge species were recorded inside the bored cavities. In addition, the species Cliona janitrix is here reported, for the first time, into archaeological artifacts. Traces of the green alga Ostreobium quekettii (ichnospecies Ichnoreticulina elegans) were found to dominate in all samples; traces of three species of microfungi were abundant. Microscopic pitting patterns and resin casts produced by different boring sponges into marble samples have been examined. Different kinds of micro-erosion are characterized by various types of ornamentation and sculpturing patterns. The data confirm that endolithic chlorophytes and fungi together with macroboring organisms, especially boring sponges, play a significant role in biodeterioration processes of submerged lapideous artifacts.

Marine boring bivalves responsible for bioerosion processes of lapideous artefacts, recovered in the Gulf of Pozzuoli (ancient Puteoli) and in the Blue Grotto in Capri Island (Naples, Italy), were examined. Intense bioerosion was observed in marble statues, limestone pavements and calcareous artefacts. Mytilid Lithophaga lithophaga, venerid Petricola lithophaga and gastrochaenid Rocellaria dubia have been identified; further two mere nestling species, arcid Barbatia barbata and trapezid Coralliophaga lithophagella, have been found inside vacant boreholes. Bivalve bioerosion plays an essential role in the deterioration of archaeological remains. Depending on their chemical composition, they are susceptible to corrosion. The determination of the damage level is very important to characterize the gravity of the biodeterioration and to define future strategies of conservation.

Endolithic microorganisms responsible for micro-erosion of submerged archaeological artifacts, found in the Underwater Archaeological Park of Baia (Naples) were analyzed. SEM observations show that the process of bioerosion involves carbonate substrates related to endolithic activity by chemotrophic and phototrophic microflorabiota. Embedding casting procedure with polyester resins permitted the identification of five different micro-borer traces, corresponding to two species of cyanobacteria, one species of green algae, one species of fungi and to a clionaid sponge (ichnogenus Entobia ). Traces of the green alga Ostreobium quekettii were found to dominate in all samples. The data confirm that endolithic cyanobacteria, chlorophytes and fungi together with macroboring organisms due to the growth of clionaids and bivalves play a significant role in biodeterioration processes of submerged calcareous artifacts.

Within the framework of the project Restoring Underwater started in 2001, the ISCR Marine Biology sector with the ISCR Underwater Archaeology Operation Unit has begun a study of the deterioration of stone artefacts exposed to marine environments (Torre Astura – Nettuno; Baiae – Naples). These studies have allowed to better understanding the factor of degradation by biological, mineralogical, and petrographic analyses and to develop measures for the protection in situ of cultural heritage. Based on what was recorded, usually the bioerosion presents various degrees of gravity: limited and sporadic damage or very serious alterations. Depending on their chemical composition, the artefacts proved to be particularly susceptible to the action of corrosion exercised by perforating animal and plant organisms.This phenomenon becomes more significant in the case in which the artefacts remain exposed and in the same position for a long period of time. It is possible to confirm that the combined action of atta...

The present study describes the role of the bivalve Rocellaria dubia Pennant, 1777 in the bioerosion process of submerged limestone artificial panels. The research was carried out in the central Mediterranean sea, in the Underwater Archaeological Park of Baiae (Naples, Italy). During the three year experimental period, the panels were submerged at 5 m depth and sequentially removed after 12, 24 and 36 months. R . dubia boreholes strongly affected stone material. The rate of bioerosion increased over time. After 36 months settlement still occurred with hundreds of boreholes and the shells were up to 13 mm in length. The results obtained concerning the impact of R . dubia on the experimental panels help to evaluate the bioerosive role of this species on submerged calcareous archaeological structures.

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.

The distribution and amount of biological colonisation on the “Lungotevere” walls (Rome) were analysed to assess the ecological role of the main environmental parameters (solar irradiation, rain winds, prevailing winds) and of the nearby tree covering. The floral data indicate a close similarity of species in the stations examined and a dominance of Chroococcus lithophilus Ercegovic among the cyanobacteria and of Erigeron karvinskianus DC among the vascular plants. In these very hard conditions the exposure and amount of the water input do not seem to influence the flora qualitatively as much as it does quantitatively. The low value of microflora, as found in areas characterised by the presence of trees, can be referred to as resulting from the “umbrella” effect of the plants. The macroflora is less influenced by such effects due to the greater capacity of these plants to derive moisture through their roots.

Underwater archaeological sites represent assets of great value that are subjected to physical and biological stresses. The study of biological settlement on calcareous substrata is the first step to understanding the biodeterioration processes that take place on man-made structures (marble, statues, columns and other architectonic elements) in marine environments. To accomplish this objective, calcareous panels (30 × 30 × 2 cm) were immersed for 3, 6, 9 and 12 months along a north−south transect in marine protected areas of Baia (Naples). Both epilithic and endolithic assemblages were analysed: the latter was observed by scanning electron microscope after the cast-embedding technique had been used. After 1 year the epilithic community was still changing, with algae and pioneer species overgrown by encrusting organisms, and the endolithic assemblage dominated by cyanobacteria/chlorophyte traces. Changes of epilithics lead changes on endolithics. The initial phototrophic borers were replaced by more sciophilous and competitive species; moreover, the first macroboring organisms arrived after the settlement of grazers. After 12 months of immersion, the rate of bioerosion was high, the material was deeply bored and all of the limestone surface was heavily covered.

The biodeterioration of stone substrata by bryophytes (Musci and Hepaticae) is due to biogeochemical and biogeophysical mechanisms. In this work the biogeochemical damage caused by epilithic moss species, found in archaeological sites of Rome, was investigated. The cellular calcium content in specimens of Grimmia pulvinata (Hedw.) Sm. was analysed using a sequential elution technique. The analyses were carried out on moss specimens sampled from different lithotypes and in different seasons in order to measure the calcium concentration both in relation to the mineral composition of the stone and to the plant physiology. The highest calcium concentration of the extracellular exchangeable fraction was detected in the samples grown on marble and in the waterlogged specimen sampled in spring time.

Before dealing with the specific problems of aerobiological monitoring, it is necessary to explain how the biological component of air may be a potential cause of degradation in cultural artefacts.

The techniques currently available for the identification of biological aerosol can be divided into five categories: cultural techniques; direct counting through a microscope; rapid biochemical methods; immunological methods; molecular biology methods.

This chapter considers two different types of environments, churches and hypogea, that share some common characteristics: they are indoor environments, and contain a great variety of culturally and artistically interesting works (paintings, vestments, sculptures, etc.), though their function is not conservation; moreover they are usually not equipped with heating, lighting or ventilation systems that have been calibrated and controlled for a correct conservation of the works of art. For other aspects, on the contrary, churches and hypogea are decidedly different from each other, particularly if they are considered from a microclimatic point of view; for this reason, we shall analyse them separately.

Twenty-two taxa of desmids are reported from a pool near Guma Valley (Sierra Leone). Six are new to the country. A new variety of Staurastrum hystrix (S. hystrix var. granulatum) is described.