Hypogea are natural or artificial spaces located underground often of great interest from an anthropological, archeological, religious, artistic, or historic point of view. Due to their features, these environments usually present conservative problems and biological colonization could be considered as one of the main threats. The present three-year study was carried out by specialists of the Central Institute for Restoration of Rome (ICR) in the hypogeous site preserved in the Roman Archeological Museum of Positano (Positano MAR) and focused on characterizing biological alterations present on the mural paintings; setting up efficient strategies and protocols for biodeterioration control; and monitoring the efficacy of direct and indirect interventions. Patinas with different morphologies were analyzed through microscopic observations, cultural analyses and next-generation sequencing. The results proved that the alterations comprised a great variety of microorganisms forming very distinct communities, differently distributed over space and time. The main taxa represented were bacteria of phyla Pseudomonadota and Actinomycetota, fungi belonging to the genus Fusarium and Gliocladium, and algae of the genus Chlorococcum. Preservation protocols were set up considering the alterations’ composition and included the application of biocides, limiting daily temperature changes, decreasing illuminance values on painted surfaces, and the screening of natural light sources.

Preventive conservation and restoration of mural paintings in hypogean environments is a significant challenge. These types of settings are particularly difficult to manage due to their peculiar morphological and microclimatic features. Since its foundation in 1939, the Central Institute for Restoration (Istituto Centrale per il Restauro—ICR) within the Italian Ministry of Culture has been engaged in the prevention and safety of these unique cultural heritage assets. Starting from a holistic analysis of vulnerability and hazards specific to hypogean environments, this study examines the main risks and corresponding conservation strategies. Particular attention is given to the impact of residual risk on restoration decision-making, with the aim of defining logistical and operational requirements to carry out sustainable and enduring interventions in these complex settings. The compatibility and durability of restoration materials under hypogean conditions are also examined. Furthermore, thanks to funding provided by the PNRR CHANGES project, a few research directions are proposed to address unresolved issues through the investigation and assessment of innovative products and methodologies. This review aims to lay the foundation for the development of guidelines incorporating protocols for the conservation and restoration of mural paintings in hypogean contexts, with particular attention to the needs and constraints imposed by these specific environments.

The frescoed Annunziata Oratory chapel in Riofreddo (Italy), a unique testimony to the pontificate of Martin V, sheds light on the trade routes of Ninfa in the first half of the 15th century. Despite having undergone several restorations in the past (the most recent in the 2010s), the Oratory presents serious conservation issues. At first glance, there are no evident signs of biological colonization; rather, the most obvious damage is attributed to detachments and saline efflorescence. Biological colonization at several points was identified using various diagnostic field and laboratory techniques such as ATPase point analysis, field stereoscopy in visible and UV light, culture-based and molecular approaches, Raman spectroscopy, and SEM analysis, biological colonization at several points was identified. The characterization of salt efflorescence was carried out using ion chromatography analysis. The presence of bacteria, fungi and algae, which are also linked to saline efflorescence, was observed. A clear correlation between the biological colonization and salt efflorescence composition was highlighted by our results, as well as the potential sources of microorganisms and salts via the capillary rise of groundwater. This early diagnostic approach regarding the presence of lithobionts and salt efflorescence demonstrates the complex interplay between environmental factors and microbial colonization, which can lead to biodeterioration processes.