NATIONAL PHD IN HERITAGE SCIENCE

Thesis title: Investigations on the Bioprotective/Biodeteriorative Dual Role of Subaerial Biofilms on Built Heritage Surfaces

For decades, biocolonization on built heritage surfaces has been regarded primarily as a threat to be controlled, and its removal has often been driven solely by aesthetic concerns. However, forefront research has begun to challenge this paradigm, suggesting that subaerial biofilms (SABs) may also play a protective role under specific conditions. This emerging perspective has sparked a vibrant scientific debate, highlighting the urgent need for targeted research to clarify the dual role of SABs in heritage conservation. Despite this growing interest, only a limited number of studies have systematically explored the interactions between SABs and porous substrates, and most existing works suffer from significant methodological constraints. This emerging yet fragmented picture underscores the urgent need for a comprehensive and interdisciplinary framework capable of linking SABs’ morphology, composition, and functionality to substrate properties and environmental conditions. Addressing these gaps requires the adaptation and optimization of currently used methodologies, many of which are unsuitable for capturing the complexity of living, dynamic interfaces such as biocolonized heritage substrates. This doctoral research aims to advance the understanding of the complex biogechemical and biogeophysical interactions between SABs and porous substrates and to assess their implications for conservation strategies. In particular, the project focuses on the adaptation and optimization of methods traditionally applied to porous materials, repurposing them for biocolonized systems, where the presence of the SAB introduces additional complexity and dynamism. The main goal was not only to measure but to understand at what level the SAB alters the atmosphere-substrate interface - affecting, for instance, gas exchange, liquid transport, and surface mechanical behavior. To address this multifaceted challenge, the research adopted a multidisciplinary and context-sensitive approach, bridging microbiology, material science, physics, chemistry, and conservation science, and integrating laboratory-based experimentation, on-site investigations, and environmental assessment tools into a unified methodological framework. The starting point was the optimization of the laboratory protocol originally proposed by Villa et al. (2015) (doi: 10.3389/fmicb.2015.01251), to grow mono-species and dual-species SABs on Lecce stone specimens, since the original protocol was not designed to capture the complexity of SAB–substrate interactions from a materials science perspective. This phase was crucial for developing reproducible and structurally mature model system SABs, providing a controlled platform for the systematic study of the SAB-substrate system. Although the laboratory setup represents a simplification compared to the complexity of natural communities, the selected dual-species configuration – combining a photoautotrophic cyanobacterium and a chemoorganotrophic bacterium – ensures a reasonable representation of real systems. This choice introduces essential ecological interactions, such as cross-feeding and spatial structuring, while maintaining experimental tractability. The optimized biocolonization protocol yielded homogeneous and adherent biofilms that enabled the reproducible, quantitative, and physically meaningful evaluation of SABs’ impact on both water-transport and surface micromechanical properties, establishing the methodological foundation for subsequent laboratory and on-site analyses. Given the pivotal role of water as one of the main agents of deterioration in porous materials, understanding how the presence of a SAB modifies water transport and moisture exchange processes becomes essential for evaluating the long-term durability of heritage substrates. Addressing these aspects was therefore a central focus of this research. Water-transport properties were investigated using a suite of complementary and refined techniques, including capillary water absorption, drying rate, hygroscopic sorption, water vapor permeability, and wettability tests. These methodologies were adapted from existing standards for porous material, eventually treated with protective coatings or consolidants, and recalibrated to account for the living, dynamic nature of SABs. Results demonstrated that SABs reduce capillary water absorption and surface wettability while preserving vapor permeability, stabilizing moisture levels within the substrate. These findings suggested that SABs may have a semi-permeable action, mitigating hydraulic stress without compromising breathability. The dual-species biofilm, in particular, showed a stronger effect, likely due to its higher Extracellular Polymeric Substance (EPS) production and structural complexity. Micromechanical characterization, performed by microindentation and microscratch testing, revealed that SABs enhance near-surface hardness, stiffness, and cohesion, demonstrating that biological colonization can locally reinforce the stone matrix. These results indicate that SABs contribute to a subtle but measurable surface hardening, possibly due to the physical presence of the microbial and EPS layer, EPS-mediated grain bridging or biocalcite precipitation. Such micro-scale stabilization mechanisms, though previously suggested in the literature, had not been quantitatively validated through comparable datasets. Together, these findings point to a potential bioprotective role of SABs in mitigating superficial mechanical wear, reducing granular loss, and stabilizing the outermost layer of the material. These laboratory findings were complemented by the case study of Palazzo Rocca Costaguta (Chiavari, Italy), where on-site analysis revealed that the SAB did not damage the plaster surface and contributed to reduced wettability. In addition, a Life Cycle Assessment (LCA) of three biocidal treatments, compared with a no-treatment scenario, further highlighted that non-intervention may be the most sustainable option when SABs do not exert detrimental effects. This research advocates for a paradigm shift in the built heritage conservation, recognizing SABs not only as agents of deterioration but also as potential contributors to material protection. The results contribute to a growing body of evidence showing that microbial colonization cannot be interpreted solely as damage, but as part of a broader equilibrium between materials, environment, and microorganisms. The methodologies adapted here open promising directions for future studies, where SABs may be evaluated not as failures of preservation but as integral components of the monument value. If demonstrated to exert neutral or protective functions, SABs could be integrated within sustainable conservation frameworks as Nature-based Solutions (NbS), contributing to the mitigation of water-related stress and the stabilization of surfaces under changing climatic conditions. Embracing stable, non-damaging biocolonization may thus enhance both the ecological and cultural value of heritage assets, aligning conservation with biodiversity-aware and sustainability principles. Overall, by advancing tailored methodologies and providing robust experimental evidence, this research supports a more informed and forward-looking approach to heritage conservation in the presence of SABs.