## 1 Introduction  
In conservation science, fresco deterioration is a well-documented and extensively studied issue [1, 2, 3, 4, 5, 6, 7, 8, 9]. The main conservation challenges affecting fresco paintings are largely attributable to their intrinsic material properties, such as the composition and thickness of the mortar layers (i.e., \"arricco\" and plaster) and the chemical nature of the pigments [1, 2, 10, 11].  
Deterioration phenomena in monuments typically involve both structural and superficial alterations and often manifest at the interface between the substrate and surface materials, occurring across the macro-, meso- and microscales. These processes can result from physical, chemical or biological factors, which are influenced by microclimatic conditions, the overall conservation state of the environment, and the properties of the substrate.  
Among these, salt crystallization is recognized as one of the most aggressive degradation processes affecting porous stone materials at cultural heritage sites. The growth of salt crystals generates internal mechanical stress, leading to several visible deterioration effects, including surface flaking, cracking, layer detachment, pigment alteration, efflorescence, sub-efflorescence and crust formation on the surface. These mechanisms can induce an irreversible loss of the original material [12; 13]. Salts may be present due to the original construction materials or may originate from external sources, including groundwater transported via capillary rise, depending on the pore size distribution and wetting behavior of the materials. Additionally, airborne pollution, such as particulate matter, marine aerosols, and atmospheric contaminants, can deposit on the surfaces and progressively penetrate into the porous matrix [14; 15; 16].  
In addition to their direct mechanical and chemical effects, soluble salts may also promote the colonization of specific biodeteriogenic microorganisms, including bacteria, fungi, and algae [17]. Therefore, the quantitative determination of both anions and cations is critical for assessing the chemical degradation risk and for understanding and preventing microbially induced deterioration [18].  
The scientific literature reports several data on the main biodeterioration phenomena, detailing their characterization and primary composition. Bacterial genera of the phylum Actinomycetota, Bacillota and Pseudomonas, and fungi classes including Sordariomycetes and Eurotiomycetes, are among the most frequently encountered. Despite being typically sheltered in indoor environments, mural paintings can also host photoautotrophic organisms. In fact, cyanobacteria (e.g., Cyanophyceaeae) and green algae (e.g., Trebouxiophyceaeae and diatoms) have been documented under certain conditions [9; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29].  
As previously noted [30; 31], microbial communities colonizing frescoes often display slow metabolic activity and may remain dormant for extended periods. Nevertheless, their biodeteriorative potential--including biomineralization, secretion of chelating or acidic compounds, and mechanical penetration by hyphae--remains a significant risk to the material integrity of artworks [20; 31; 32].  
This study aimed to identify biological colonization affecting frescoes that are not visible to the naked eye and to assess the potential correlation between characterized salt efflorescence and microbial growth on the painted surfaces of the Annumziata Oratory (Riofreddo, Italy), through a multidisciplinary diagnostic campaign. The investigation began with non-invasive in situ analyses conducted with the support of MOLAB, the mobile laboratory of the E-RIHS research infrastructure ([[https://www.e-rihs.it/en/laboratori-mobili/](https://www.e-rihs.it/en/laboratori-mobili/)]([https://www.e-rihs.it/en/laboratori-mobili/](https://www.e-rihs.it/en/laboratori-mobili/)), accessed 30 March 2025), to determine the main lithobiants and soluble salts involved aimed to a correct conservation strategy.