Microorganisms in cultural heritage. Study and development of new green techniques

This doctoral thesis was funded by the PON Research and Innovation program, which in 2021 promoted scientific research on green topics. Within this framework, the research developed is characterized by many interconnected macro-themes. The underlying assumption is that the microorganisms present on cultural heritage, which in specific conditions cause biodeterioration, can also be sources of new, more sustainable and green substances for conservation and restoration. This idea enabled the development of a thesis with an almost circular structure, where the biodeterioration of the artworks is studied in detail, microorganisms are isolated, and studied for potential biotechnological applications. Microorganisms are present everywhere on Earth. Once they colonise an object, they interact with it creating a specific microbiome. Just as humans, animals, and plants have their own microbiomes, so does cultural heritage, both organic and inorganic. Inorganic cultural heritage, such as lithic surfaces, are often studied since microorganisms that live on them have developed specific metabolic processes to ensure their survival. Hypogeal environments are characterised both by lithic surfaces and specific environmental features promoting the proliferation of microorganisms, which can establish a very specific microbiome. For these reasons, the conservation of hypogeal environments is a worldwide problem which increase in complexity when precious mural paintings are present inside. In particular, dark-pigmented fungi have gained great interest in recent decades due to several uncontrolled proliferation cases. This research has been carried out using a multi-analytical and multi-omics approach highlighting the relevance of these techniques in cultural heritage studies. The chapters of this thesis show the experiments performed to support the work of archaeologist and restorers, who needed a microbiological support by conservation scientists. Chapter 1 of the thesis outlines the fundamental topics found in the literature and provides the theoretical basis for the entire work conducted in the following chapters while chapter 2 clearly defines the general and specific objectives of the work performed. In chapter 3, the microbial communities present on the mural paintings inside two hypogeal tombs of Monterozzi necropolis of Tarquinia (Rome, Italy) have been studied. This represents the first essential step to better understand these realities. The pictorial layer of both tombs is characterised by black spots randomly distributed which have been sampled through different tools. The components of the microbial communities have been isolated and studied through culturable-dependent and metabarcoding analysis. The results obtained show many similarities between the fungal microbial communities of the case studies here reported and the main European hypogeal environments such as Lascaux cave. To better understand the role of MOs in black spots formation, in chapter 4, the metabolic activities and mechanisms of these microbial communities have been analysed in detail. The analyses have been performed using a multi-analytical approach. Besides more common microbiological assays and analytical techniques, such as Raman spectroscopy and SEM-EDX, an innovative proteomic approach has been used to clarify the metabolic pathways for melanin production. The results obtained pointed out the low efficiency of traditional biocides in block the growth of fungal strains, the presence of fungal strains able to precipitate calcium carbonate and the overexpression of specific proteins for some dark-pigmented fungi. In chapter 5, instead, the beneficial properties of a bacterial strain isolated from a canvas have been studied. Using a classical microbiology approach and proteomic analysis, the antimycotic properties of the bacterial strain have been studied in detail to explore a possible new eco-friendly alternative. The untargeted proteomic approach was adopted to study the entire proteome of the single strain culture and the co-culture of bacterial and fungal strains. From these results, the production of enzymes, antibiotic resistance proteins and non-ribosomal peptide was pointed out. Considering the episodes of massive proliferation experienced in other hypogeal environments, the approach adopted in the case of the tombs of Monterozzi necropolis of Tarquinia provides a virtuous example of how preventive measures can effectively identify problems at an early stage. Furthermore, whenever direct control would be necessary in this site in the future, the preliminary but promising results regarding the ability of bacterial strain to block fungi have paved the way for a new applications. This work is considered a starting point for further studies into the complex world of hypogeal environments in order to offer both the proper and more advanced methodology to prevent hazardousness proliferation and to preserve precious artworks.