Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts

www.nature.com/scientificreports OPEN Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts Maria Stratigaki 1*, Andrea Armirotti 2, Giuliana Ottonello 2, Sabrina Manente 3 & Arianna Traviglia 1 Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC–MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper’s surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans. Handwritten codices, documents, and letters are valuable cultural heritage materials exhibiting exquisite artisanry and possessing immense value as they preserve, convey, and communicate centuries-old written information across extended periods of time. Their paper substrates, however, suffer from natural a geing1,2, which significantly undermines their long-term stability. Numerous factors contribute to the degradation of paper, and these factors can be categorised according to their nature. Physicochemical factors include light, temperature, relative humidity, pH, microclimate, storage conditions, hydrolysis and oxidation, composition, raw materials, fillers and additives3–6. Mechanical factors cause wear and tear a ccumulation7, while biological factors arise from the colonisation of microorganisms, primarily fungi and bacteria, producing corrosive metabolites8,9. The degradation processes caused by these factors render paper-based writing supports fragile and vulnerable, thereby endangering their preservation for the future. The biodeterioration of writing substrates is the result of intricate interactions at the micro- and nano-scale, leading to discoloration, foxing, embrittlement, cracking, or, ultimately, complete loss of structural i ntegrity10–12. Certain fungal species, for example, produce metabolic waste that causes visible coloured stains on the substrate at a macro-level. Active metabolites, such as enzymes and acids, secreted by these microorganisms can further lead to the acid hydrolysis of p aper13. Yeasts, members of the Fungal Kingdom, are highly dependent on aerial spread and produce pseudo-hyphae to replicate and move to the s ubstrate14. Bacteria also pose a significant threat to paper degradation due to their strong metabolite products. These microorganisms can remain viable but in a dormant state for y ears15,16, and reactivate upon exposure to optimal conditions in a favourable environment. 1 Center for Cultural Heritage Technology (CCHT), Istituto Italiano di Tecnologia, Via Torino 155, 30172 Venice, Italy. 2Analytical Chemistry Facility, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy. 3Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Via Torino 155, 30172 Venice, Italy. *email: ; Scientific Reports | (2024) 14:7003 | https://doi.org/10.1038/s41598-024-57228-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ Microbial colonisation can thus thrive in extreme environments17,18 or conditions with elevated levels or sudden fluctuations in temperature or humidity, which accelerate the saturation of paper substrate with water, although this is not a strict requirement. The cataloguing and mapping of fungal and bacterial diversity, facilitated by different techniques for detection and identification, lays the foundation for protecting historical paper documents19. This approach prevents irreversible damage over time, allowing the public to access these documents without substantial loss. However, cultivating bacterial and fungal structures using traditional in-vitro methods can be challenging due to factors such as the selectivity of the media, temperature, and t ime20–22. To address these limitations, molecular approaches offer significant benefits in detecting the species’ fi ngerprint23. Omics techniques are increasingly used in the field of cultural heritage24,25 to understand the biodegradation mechanisms of microbial communities and assess biodeterioration levels. Proteomics, in particular, can analyse residual trace proteomes discovered on manuscript pages, providing valuable insight into the health conditions or causes of death of the w riters26,27. The use of traditional imaging methods, like optical microscopy (OM) and scanning electron microscopy (SEM), to locate species on the surface of the paper fibre network is prevalent in this field. However, to improve our understanding of intricate and complex biodegradation p rocesses28, it is essential to visualise microorganisms beyond and beneath this surface. Here, we introduce a novel methodology in the field of cultural heritage and historical paper biodeterioration. Our approach utilises fluorescence microscopy as a non-invasive and non-destructive imaging technique. The method relies on the natural intrinsic autofluorescence of fungal and bacterial cells, resulting from endogenous fl uorophores29, instead of externally integrated fluorescent m arkers30,31. Although the autofluorescence of these species is well-documented32–34, only a few reports have investigated this possibility in the field. This study focused on unravelling the microbial threats posed to seventeenth century historical handwritten letters, stored in the Library of the Correr Museum (Biblioteca del Museo Correr) in Venice. These letters originate from Veneto, Italy, a region renowned for its long tradition and rich history in papermaking35. To conduct an in-depth investigation and evaluation of the microbial presence and diversity, a synergistic approach was adopted36–38. To reveal and assess individual features (...truncated)