Abstract
Abstract Cultural heritage, according to UNESCO’s definition, includes monuments, sites, and traditions that testify historical, artistic, and social values of humanity. This broad and multifaceted concept encompasses three main dimensions: tangible, intangible, and natural heritage. Tangible cultural heritage includes objects and buildings that represent human history and creativity; intangible heritage, by contrast, refers to traditions, rituals, and performing arts that support cultural diversity in an increasingly globalized world. Finally, natural heritage comprises landscapes, geological formations, and ecosystems of exceptional scientific, aesthetic, or ecological value. Together, these three dimensions constitute a unique and non-renewable resource, essential for collective identity and for the social and economic well-being of communities. Its protection therefore represents a global priority. The great variety of cultural heritage, however, also makes it vulnerable to an equally diverse range of threats: climate change, pollution, mass tourism, and biological colonization are just a few of the pressures that compromise its integrity. Its protection requires a multidisciplinary approach, at the heart of heritage science, a field dedicated to the sustainable conservation and transmission of this invaluable resource to future generations. Within this context, tangible heritage plays a particularly significant role. Materials such as stone, wood, and parchment are constantly exposed to environmental factors that threaten their preservation. Even stone, one of the oldest and most durable building materials, is subjected to a slow deterioration process under the action of atmospheric pollutants, acid rain, and thermal fluctuations. These abiotic factors often promote biological colonization by bacteria, fungi, algae, and lichens, which in turn accelerate decay processes. Microorganisms growing on stone surfaces frequently form subaerial biofilms (SABs), complex microbial communities embedded in an extracellular matrix. These biofilms appear as colored patinas, whose pigmentation reflects their metabolic activity. Although biofilms are generally associated with biodeterioration phenomena, in some cases they can exert a protective effect by stabilizing surfaces or promoting the formation of mineral coatings that preserve the substrate. Their impact depends on multiple environmental variables and on the nature of the material itself; hence, accurately assessing their role is essential for designing effective conservation strategies. On the other hand, sites of natural heritage hold immense ecological, cultural, and economic value, but they also represent some of the planet’s most fragile ecosystems. Their conservation requires a dynamic balance between human activity and environmental preservation. Maintaining the original ecological conditions of these sites is crucial, as ecosystems are the results of long coevolutionary processes among the species that inhabit them. Recent studies have also highlighted the crucial role of microbial communities in ecological stability, nutrient cycling, and environmental resilience. Understanding these microscopic dynamics—through the use of advanced molecular tools—is now recognized as a key element in the protection of natural heritage. Most research on microbial diversity in cultural heritage focuses either on DNA-based metabarcoding to identify microbial taxa or on cultivation-based physiological studies. While metabarcoding reveals community composition, it cannot distinguish metabolically active microbes. Conversely, laboratory cultivation provides physiological information but does not accurately replicate natural conditions. This limitation is particularly pronounced in natural heritage, where molecular techniques are underutilized. Current conservation research is increasingly moving towards a holistic approach, integrating the study of microbial communities with abiotic factors like substrate properties and microclimate. However, most studies still focus on composition rather than functional activity. Multidisciplinary approaches that combine molecular tools with environmental analyses could improve predictive models of microbial activity and inform sustainable, safe conservation strategies. This thesis investigates the use of molecular techniques as innovative tools for developing models to conserve both stone-built and natural cultural heritage. Through three case studies, it applies distinct molecular approaches to understand how microbial communities interact with different substrates and their surrounding environments. The first study (Chapter 3) investigates fungal colonization in two gypsum crusts from the Galápagos Islands using a DNA metabarcoding approach. The results show that the chemical and mineralogical properties of the substrate strongly influence the structure and activity of fungal communities. Colonization is not a stochastic process but rather the result of complex interactions between microorganisms and microenvironmental factors that select for specific metabolic traits. Understanding these relationships is therefore crucial for heritage conservation, as it reveals how microbial ecology and material composition can determine processes of biodeterioration or bioprotection. The second study (Chapter 4) employs mRNA sequencing (metatranscriptomics) to examine four differently colored subaerial biofilms on the wall of a chapel. Its goal is to assess whether biofilm color can discriminate distinct metabolic activities and taxonomic compositions. The findings demonstrate that each color corresponds to a unique metabolic and taxonomic profile, and ecological strategy: color, therefore, is not merely an aesthetic feature but a visible manifestation of microbial activity. Pigment analyses further confirm this correlation, suggesting that biofilm coloration can serve as a non invasive bioindicator of the conservation state of stone surfaces. Although preliminary, this study proposes color as a rapid and sustainable diagnostic tool for monitoring materials. The third study (Chapter 5) extends the temporal perspective by using sedimentary ancient DNA (sedaDNA) to explore past ecological dynamics. SedaDNA analyses revealed the co-occurrence of ancient plants and microorganisms, suggesting deep coevolutionary relationships, particularly between plants and nitrogen-fixing bacteria. The results indicate that ancient boreal soils functioned as complex ecosystems analogous to modern Arctic environments. This demonstrates the potential of ancient DNA for reconstructing past ecosystems, identifying mechanisms of ecological resilience, and guiding conservation strategies aimed at functional ecosystem restoration rather than static preservation. Overall, this thesis integrates DNA-, RNA-, and ancient DNA-based methods to provide a more comprehensive view of microbial diversity and activity, capturing not only “who is there,” but also “what they are doing,” and “what they were.” Combining molecular data with environmental and material analyses, these approaches can open new possibilities for sustainable, eco-friendly conservation practices that consider both the biological and ecological dimensions of cultural heritage preservation.