The common purple damage of ancient parchments has been studied by comparing damaged and undamaged areas from the same document (the roll named A.A. Arm. I-XVIII 3328). An integrated multidisciplinary approach was adopted, making use of molecular, chemical and physical techniques. To the best of our knowledge these techniques have never been used in combination to analyze the purple spots damage of parchments.

This multidisciplinary approach yielded a great amount of interesting information, useful to decipher what the parchment suffered during its 800 years-long story. The new Light Transmitted Analysis (LTA) technique allows to study and localize the structural damages without introducing any bias caused by the sample preparation: in the purple spots, it showed that the alteration mainly affects the collagen matrix, saving the larger fibers. The Raman specter analysis provided clues about the bacterial pigments at the basis of the purple discoloration, detecting microbial rhodopsins: the well-known archaeal bacteriorhodopsin19 and/or the more recently discovered proteorhodopsins20, which are largely diffused in aerobic marine bacteria21,22,23. The Next Generation Sequencing (NGS) approach, yields a much deeper resolution of complex microbiota than other molecular methods. The primer pair used in this study (515F/806R, employed in the Earth Microbiome Project; www.earthmicrobiome.org) allow the identification of a wide range or microorganisms. Although Hugerth et al.24 pointed out that these primers miss some archaeal phyla, a test performed with the Probe Match tool in RDP25 revealed that they match the Euryarchaeota (515F = 0.95; 806R = 0.95) and even better the Halobacteria, which were actually looked for, according to the Raman results (515F = 0.97 and 806R = 0.96). As expected, the NGS output highlighted significantly different communities in the two sets of samples. The taxonomical assignation of the OTUs adds even more information, depicting the relative frequencies of the different bacterial phyla and orders. Moreover, a BLAST analysis revealed that OTUs find their best homologues within both cultured and uncultured microorganisms from environmental samples (mainly salt-related or soil ones), and human (mainly skin-associated). A detailed description of the best homologies for the OTUs occurring more than five times is shown in Supporting Table S2.

Evidences linking bacteria to the degradation process include: (i) some rhodopsins-producing microorganisms, responsible of the purple discoloration; (ii) the damage of the parchment surface linked to rarefaction and/or disappearance of the collagen matrix; (iii) the microbial community structure, actually different in the damaged and undamaged portions of the parchment; i.e. Actinobacteria, mainly Pseudonocardiales, dominant in the undamaged areas of the parchment, while Proteobacteria, mainly Gamma, with the prevalence of Vibrionales, prevailing in the purple spots, although Actinobacteria were also abundant.

As a whole, these results can be explained in the framework of a successional model.

A conspiracy in several acts: a microbial succession is the model of colonization

Parchments are made from animal skins, more precisely they consist of the dermal skin layer only. The procedure for their preparation have remained the same in the course of the centuries. Since ancient times, to reach the final thickness and smoothness, skin was subjected to a series of treatments to prevent putrefaction (sea salt, sodium chloride), to remove the hair (lime, calcium hydroxide), to allow ink attachment, and for the whitening and/or smoothening of the surface26. The salt treatment seems not have been usually used in the northern regions27.

In the Southern coastal regions (e.g. Italy) or whenever it was cheap and available, sea salt was used to preserve the hides, until processed, soon after the animal were slaughter2, 28, 29. Salting was carried out dry or in tanks, where the skins were plunged in brine for some days30 so that the salt ions entered deeply into the skin. It is easy to suppose that the brine could also act as a culture/enrichment medium for salt carried halophilic and halotolerant microorganisms (archaea, marine bacteria and their resting phases), in ancient times surely present in the marine salt. After liming and soaking in water, the entire skin was then placed on a stretching frame, deeply scraped and shaved until the thin final feature was obtained. Hence, it is possible to hypothesize that at the end of the production procedures, the parchment internal environment was salty, with a gradient of decreasing salinity from the outer layers of the animal skin inwards. In this environmental conditions, halophilic archaea and halotolerant bacteria, located into the reticular collagen structure, were both preserved.

Once in the monasteries, the parchments were stored in the armaria 31 along the cloisters walls, exposed to moisture, changing temperature, and light, whenever they were unrolled in the reading rooms. Hence, parchment environment probably changed with time and local events, enabling the growth of the microbial colonizers, which were already present in the parchment. As regard the A.A. Arm. I-XVIII 3328 parchment, it belongs to the oldest collection of the Archives, called Fondo “Archivum Arcis”, which was kept in Castel S. Angelo (downtown Rome) until the end of the XVIII Century. In ancient times Castel S. Angelo was exposed to frequent and important floods of the Tiber River32 which could have reached the library and wet the roll.

Molecular methods reveal DNAs, which have not been removed in the production processes or later destroyed, but they do not obviously tell us the order of the events they witness. So we hypothesized a succession models, taking into account the possible provenience of the detected microorganisms, the selective forces allowing them to root, and the biological role they could have played. According to this information and to our results, the model of parchment deterioration is a microbial succession acting in two main phases (Fig. 7).

Figure 7 The two-phase model of parchment colonization. The microbes involved in the parchment damage are: halophilic Archaea (purple, the cells are intact in the 1st phase draw or collapsed in the 2nd one); Gamma-Proteobacteria (gold) and Fungi (teal). Full size image

Abiotic and/or biotic agents can be considered as causative agent of the purple spot damage of the parchment, but the Raman analysis results point to the biotic ones. In this last case, a common initial colonization phase of the purple-damaged parchments can be hypothesized, having two possible scenarios, which could have been intertwined, occurring at different times or, even simultaneously, in different areas in the same parchment.

In fact, the microbial rhodopsins detected by the Raman spectrum, could originate from halophilic Archaea (Halobacteria) or from halotolerant Bacteria (probably Proteobacteria). A simpler hypothesis relies on proteorhodopsin-producing Bacteria, slowly growing in the nutrient poor parchment, staining and degrading it in several waves. Moisture, light, low temperatures and scarce availability of nutrients, all of these inducing the proteorhodopsin production could have been the triggering factors. However, the salty environment of the inner part of the dry parchment should have rather fostered the Halobacteria, dominant colonizers of the evaporation ponds and surely present in the marine salt used for the brining. With this in mind, Halobacteria should have been firstly acting as the main character in a more complex, two steps scheme, similarly to what happens nowadays in the red heat deterioration of brine-cured hides33. In ancient times, when parchment started to be exposed to humidity and light, only Halobacteria were allowed to grow in a sub-superficial layer of the parchment at the flesh side, where environmental conditions are permissive to them: Halobacteria need at least 5–10% NaCl for their very existence, take the dominance in low nutrient contexts, and like warm temperatures34. When the parchment moistened due to local events, probably in the warm season, the Halobacteria, associated to salt particles inside the parchment, began to grow in the still intact structure, starting to deteriorate the collagen matrix. The bacteriorhodopsin producing colonies formed the core of the purple spots and haloarchaeal collagenases enriched the available nutrients in the early spots. Although the 515F/806R primers have been proven suitable for the amplification of Halobacteria, most of which produce bacteriorhodopsin, no related amplicons have been observed in this as in other works2; however, Halobacteria lyse whenever the salt concentration lowers below the limiting value, because the envelope glycoproteins need a high concentration of NaCl for their structural stability. Hence, the released archaeal cellular content could have provided further nutrients to the fast-growing Gammaproteobacteria which, besides going on in consuming the collagen matrix, wiped away the halobacterial debris, only leaving the purple stain. This could explain why the NSG approach failed to detect Halobacteria. In a nutrient-rich environment, the fast growing marine Gammaproteobacteria, which are adapted to a salt concentration of about 3%, expand, and outcome Halobacteria, especially at low temperatures34. This should have been occurring in the cold season and whenever the water content inside the parchment exceeded 15%35. The microbial growth in neighboring areas, at the same sub-superficial level, can be responsible of the high fragility of the surface layer of the parchment and the loss of the written areas on the flesh side (Fig. 2c).

In the second phase, the colonizers identity links to the individual history of each parchment. As time passed by, other environmental factors had been intervening: dust settled on the parchment surface, bringing many new bacterial intruders, so as did the repeated human handling.

Regarding the A.A. Arm. I-XVIII 3328, the second phase dominant OTUs are Actinobacteria, prevalently unclassified Pseudonocardiales or Saccharopolyspora; on other documents2, the dominant OTUs were Actinobacteria or Firmicutes, alone, together or mixed with Gamma-Proteobacteria, according to the document. Different second phase colonizers were found in the history of the Archimedes’ palimpsest:36 Gammaproteobacteria dominated and some accident involving a eutrophic freshwater environment seems to have been occurred.

Still, the parchment itself keeps on in exerting a selective pressure, so that, for example, Actinobacteria are always mainly represented by Pseudonocardiales, whilst Streptomycetes, among the most common soil microorganisms, are present neither in our parchment nor in the other two described in the literature. Among the man derived microorganism, many are typically associated to the harsh skin environment and able to stand low moisture and/or fairly high salt concentrations (e.g. micrococci, staphylococci, Acinetobacter).

Of course, even in the same document, the already damaged areas offer different environments: the NGS results, indeed, show that the distribution within Pseudonocardiales is not even. OTU #1 is more than 80 folds more frequent in the damaged areas, whilst OTU #2 about tenfold in the undamaged ones. Interestingly, the best homologues to OTU #1 are some out of the uncultured Saccharopolyspora-like sequences found by Pinar2 as shown in supporting material (Table S2); so, a particular group of Pseudonocardiales could actually find a suitable niche in the modified environment of the purple damaged areas. The best scoring homologue to OTU #2 is the Saccharopolyspora sp. AFM 10238 (98%, KF673492) firstly isolated from the Dead Sea.

As final considerations, at the end of the colonization probably both the complexity of biological attack and the later drying of the parchment cooperated to the cracking, detachment and loss of superficial pieces at the flesh side of the parchment, including the written areas. Furthermore, in the second phase, often fungi worsened the damage; fungi are also linked to the individual history of the parchment, and it should be interesting to investigate how they were selected by the changing environments of the damaged/undamaged areas of the parchment.

In conclusion, the integrated approach including physical, chemical and microbiological tools, has been able to give a detailed picture of the processes and actors of the bacterial alterations found on the ancient parchment. The NGS is the most affordable tool to appreciate also the fine-tuned differences in the quality and quantity of the past and present microbial colonizers. The Raman analysis offers a precious clue for the actual identity of the microbial pigments in the purple spots, recalling - on a scientific basis - the analogy with the red heats affecting today’s hides. The new Light Transmitted Analysis technique allowed to assess the amount of damages affecting the structure of the collagen matrix.

The new information on the colonization and deterioration processes, including the kind of damage to the collagen fibers and the chemical composition of the purple spots, open new perspectives to the restoration and conservation of ancient damaged parchments. The empirical procedures of conservation currently in use (controlled atmosphere and moisture, protection from light) are completely sound to avoid new damages to the ancient documents, but further studies could help to understand which kind of rhodopsins are involved in the purple spots to look for possible new restoration approaches for damaged documents. Moreover, a better understanding of the possible role of Halobacteria could be useful, as they can survive for huge times37, being a possible “time bomb” in the ancient undamaged parchments.