An experimental study was conducted to assess the nature and extent of the biodeterioration of the limestone in the Batalha Monastery in Portugal. Stone fragments covered with microbial biofilms and lichenous crusts were investigated using Optical Microscopy (OM), Low Vacuum Scanning Electron Microscopy with Energy Dispersive Spectroscopy (LV-SEM + EDS), and X-ray micro-Diffractometry (μ-XRD). Microbial samples were collected from the stone surface, cultured, and analyzed with NGS metagenomic DNA test to classify the bacterial communities associated with the formation of the biofilms. Particulate air pollutants collected on Pall GN-6 paper filters using a cascade impactor were characterized by SEM-EDS + NGS. The results showed that lichens play a major role in biodeterioration by promoting both physical and chemical attack on the limestone substrate via hyphae mechanical penetration along calcite inter-crystalline spaces, the dissolution/leaching of calcite minerals, and the precipitation of secondary minerals such as Ca-oxalates within the stone porosity framework. DNA analyses identified the bacterial communities within the biofilms and their relative abundances. Air quality monitoring results suggest that the microbial population colonizing the monastery limestone could at least partially be derived from the dry and wet deposition of airborne biological particles on the stone surfaces and that S, N, and P-rich air pollutants may have provided nutrients and energy for the bacteria communities, thus indirectly facilitating biofilm formation, the growth of a lichenous crusts, and limestone biodeterioration effects.

Pristine environments, such as caves, are unique habitats that are isolated from human activity and are exposed to extreme environmental conditions. These environments are rich sources of microbial diversity, and the microorganisms that thrive in these conditions have developed unique survival skills. One such skill is the biosynthesis of secondary metabolites with potential bioactivities, which provide the organisms with a competitive advantage in these extreme environments. The isolation and characterization of microbial strains from the surfaces of pristine cave environments are important for exploring the biotechnological potential of these organisms. These studies can reveal new products with antibacterial, antifungal, anti-inflammatory, antioxidant, and anticancer activities, among others. In addition, the identification of specific compounds responsible for these biological activities can contribute to the development of new drugs and products for sustainable biotechnological applications. Recent developments in genomics, bioinformatics, chemoinformatics, metabolic engineering, and synthetic biology have opened new possibilities for drug discovery, making the exploration of bacterial secondary metabolites more promising. In recent years, several bacteria with bioactive potential have been described, and several compounds with bioactivity have been identified. These findings are essential for the development of new drugs and products for the benefit of society. This paper discusses the potential of microorganisms found in pristine cave surfaces as a source of new metabolites with bioactivity that could have sustainable biotechnological applications. The authors suggest that more research should be conducted in these environments to better understand the microorganisms and the biosynthesis of these metabolites and to identify new compounds and metabolic pathways that could be of interest for the development of new drugs and products. The aim is to highlight the importance of these habitats as a potential source of new bioactive compounds that could be used for sustainable biotechnological applications.