# Metagenomic and microbiological analyses of historical manuscripts for bacterial community profiling and bacteria-related biodeterioration assessment

## TL;DR

This study uses modern techniques to analyze bacteria in historical manuscripts, revealing their role in damage and offering insights into their history and materials.

## Contribution

The study combines metagenomics and microbiology to uncover bacterial communities and historical handling in manuscripts.

## Key findings

- Metagenomic analysis revealed a diverse bacterial community, including cellulolytic and proteolytic genera linked to paper deterioration.
- High abundances of gut and human-related bacteria indicated extensive historical human handling of the manuscripts.
- The detection of Pseudonocardia broussonetiae suggests the use of paper mulberry in one manuscript, shedding light on Islamic paper production.

## Abstract

Bacteria are important agents in the biodeterioration of cultural heritage objects, including historical manuscripts. Characterizing bacterial communities and generating robust microbiological data has therefore become crucial for conservation and restoration strategies. In this study, we investigated the bacterial communities associated with biodeterioration in six historical manuscripts using both culture-dependent and culture-independent (Illumina MiSeq) approaches. Culture-dependent methods yielded only 16 viable and culturable isolates, highlighting the limitations of traditional techniques. In contrast, metagenomic analysis revealed a far richer and more diverse bacterial community, capturing both living and non-living microbial traces accumulated over centuries. Bacterial genera with known cellulolytic and/or proteolytic activities, such as Bacillus, Stenotrophomonas, Pseudomonas and Acinetobacter, were identified as part of a core microbiome commonly associated with paper deterioration. High abundances of gut-associated bacteria (Prevotella, Faecalibacterium, Bacteroides, Porphyromonas) and human-related taxa (Staphylococcus, Streptococcus, Cutibacterium) indicated extensive historical human handling. A notable finding was the detection of Pseudonocardia broussonetiae, an endophytic bacterium associated with paper mulberry (Broussonetia papyrifera), suggesting the possible use of this plant as a papermaking material in one manuscript. This represents an important contribution to understanding Islamic paper production. Overall, our results demonstrate that effective conservation strategies require a detailed understanding of each manuscript’s microbial ecology, together with evidence of past environmental conditions, handling history, and production materials.