Microbial Community of an 11th Century Manuscript by Both Culture-Dependent and -Independent Approaches

In this study, the microbial community (Bacteria, Archaea, and Fungi) of a historical manuscript was investigated by microscopic observation (field emission scanning electron microscopy), culture-dependent methods, and sequencing of amplicons on the Illumina MiSeq platform. Microbial isolates were also assayed for production of hydrolytic enzymes (cellulase, amylase, and protease). Observation of gold-coated paper samples in high-vacuum mode revealed bacterial cells, conidia, and fungal mycelium among the fibers. Eleven bacterial strains, including species of Bacillus, Streptomyces, Corynebacterium, and four fungal strains, including species of Aspergillus and Alternaria, were isolated by culture-dependent methods and identified by 16S rRNA and ITS sequencing, by comparison with the EzBioCloud and MycoBank databases, respectively. Illumina MiSeq sequencing revealed the presence of 20 bacterial genera, including Pseudomonas, Bacillus, Citrococcus, Promicromonospora, Carnobacterium, Arthrobacter, Salinibacterium, and Streptomyces. According to the number of reads, Pseudomonas (58%) was the most abundant genus and P. stutzeri (55.2%) was the most abundant bacterial species. Sequencing results of fungal amplicons indicated 62 genera, including Penicillium, Aspergillus, Alternaria, Mucor, and Chaetomium. Penicillium (58.89%) and Aspergillus (33.02%) were the most abundant genera. P. polonicum (55.45%) and A. ruber (22.55%) were the most abundant species of the fungal community. The results of searching for archaea in the microbial community of the manuscript by culture-dependent and high-throughput amplicon sequencing were negative. The enzymatic activity of microbial isolates indicated a potential risk of biodeterioration. It seems that using a combination of culture-dependent and high-throughput sequencing methods has a significant advantage over using them individually.