A multiphasic approach for investigation of the microbial diversity and its biodegradative abilities in historical paper and parchment documents

The microbial diversity of different kinds of stains present on the surface of 14 historical documents (nine parchments and five paper letters) was evaluated through a combination of cultural and molecular methods. The samples were recovered using adhesive tape and swabs and were afterwards treated in two different ways: (1) direct inoculation on agar plates; or (2) suspension in physiological solution and plating in specific plates for the growth of bacteria and fungi. The isolated microorganisms, before identification, were selected by two different PCR-based methods – f-ITS and f-CBH, for bacteria and fungi, respectively. The f-ITS method is based on the amplification of the internal transcribed sequence between the bacterial 16S and 23S rDNA. The f-CBH method is a new molecular selection tool oriented to the fungal cellobiohydrolase gene. Both PCR selection methods produced typical profiles, which clustered the isolates in order to reduce them for subsequent sequencing identification through the amplification of the fungal 28S rRNA and the bacterial 16S rRNA genes. The cellulolytic and proteolytic abilities were screened through the use of three plate assays, the Ostazin Brilliant Red H-3B (OBR-HEC), milk agar, and gelatin agar. Massilia timonae, Lysobacter dokdonensis, and strains belonging to the genus Bacillus sp., Microbacterium sp., and Curtobacterium sp. with different fungal members such as Aspergillus fumigatus, Penicillium commune, Mucor spinosus, and Phoma herbarum (all recovered from paper) displayed both biodegradative activities. The parchment isolates with a marked proteolytic activity were Bacillus cereus, Staphylococcus epidermidis, Pseudomonas oryzihabitans, Virgibacillus sp., Micromonospora sp., and again members of the fungal genera Penicillium, Mucor, and Phoma.

► The microbial community contaminating parchment and paper documents was investigated.
► The isolated microflora was selected through 2 fluorescence PCR assays, f-ITS and f-CBH.
► The novel f-CBH is based on the amplification of the fungal cellobiohydrolase gene.
► The 16S rDNA and the 28S rDNA sequencing identified the bacteria and fungi respectively.
► Proteolytic and cellulolytic tests showed the biodegradative abilities of isolates.