Abstract
Biological recolonization after cleaning remains a major challenge for the conservation of stone cultural heritage. As recolonization can start within months to a few years following intervention, developing rapid, field-deployable diagnostic approaches is crucial to better monitor microbial reappearance and to assess treatment performance in real time. Traditional evaluation methods lack the capacity to take into consideration non-cultivable microorganisms or assess functional traits relevant to recolonization. To bypass this gap, we applied on-site direct Whole-Genome Sequencing (Oxford Nanopore® MinION™ sequencer) coupled with colorimetric analysis to understand the microbiome, resistome, and metabolic traits of subaerial biofilms present in untreated and treated (recolonized) areas of stone statues at the “Largo da Porta Férrea” (Coimbra’s UNESCO World Heritage site). Colorimetric analysis (ΔE of 32–40 in recolonized vs. 19–43 in untreated areas) and genomic data pointed out that the applied treatment provided only a short-term effect (roughly 4–5 years), with a marked decline in fungi (1–2% vs. 7–18%), coupled with an increased recolonization mainly by Cyanobacteriota (circa 35–45%) and several stress-resistant Bacteria (globally ~95% of reads vs. 73–79% in controls). Antimicrobial resistance profiles significantly differed between sites, with treated areas showing distinct and unique resistance genes, and plasmids containing the blaTEM-116 gene, which can indicate potential adaptive shifts in the resistomes profiles after intervention. Metabolic pathways analysis revealed that untreated areas retained more complete nitrogen and sulfur cycling gene sets, whereas treated areas showed reduced biogeochemical gene contents, consistent with earlier-stage recolonization steps. Given the current recolonization detection and the ongoing biofilm formation, routine monitoring efforts (e.g., every 6 months) are recommended. Overall, this study demonstrates the first on-site genomic characterization of recolonization events on heritage stone, providing a practical prompt-warning tool for conservation monitoring and future biofilm management strategies.