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Discoveries and oddities in library materials
Microchemical Journal 124 (2016) 568–577
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Discoveries and oddities in library materials Marina Bicchieri a,⁎, Flavia Pinzari b,c,1 a b c
Laboratorio di Chimica, Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Via Milano 76, 00184 Roma, Italy Laboratorio di Biologia, Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Via Milano 76, 00184 Roma, Italy Natural History Museum of London, Department of Life Sciences, Cromwell Rd, London SW7 5BD, United Kingdom
a r t i c l e
i n f o
Article history: Received 14 July 2015 Received in revised form 28 September 2015 Accepted 28 September 2015 Available online 9 October 2015 Keywords: Raman SEM–EDS Inks Jarosite Elderberry
a b s t r a c t Surprising discoveries can happen while analyzing library materials. This paper presents four case studies focusing on four artifacts of different ages and provenances, all analyzed and studied at the Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario (Icrcpal). The common denominator amongst the case studies presented in this work, lies in the need for a strong interconnection between the analysis of biological issues and chemical data. The interplay between the two complementary approaches is paramount for the correct interpretation of the status of the material under examination as, as will be shown in the following case studies, chemical data could find an interpretation only in the face of biological observations and vice versa. The first case we here present, deals with two parchment codices that have been found in 2008 during a fishing expedition in the Canale di Sicilia. At that time a deep-sea fishing boat belonging to the fleet of Mazara del Vallo caught in its nets, two artifacts, leading to the first discovery of library materials in the deep sea. The Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS) analyses allowed for the creation of a compositional map of the surface of the samples, where several microscopic encrusting sea organisms and biogenic materials were also observed. Almost nothing of the original structure of the parchment (of which the object is presumably made) could be recognized at SEM. The absence of the typical collagen features was confirmed by Raman analyses that could only collect spectra of an isoprenic-type polymer of putative biogenic origin. The second case deals with fragments of medieval manuscripts that were found in 2007 buried in the walls of the Great Mosque of Sana'a in Yemen. An outstanding discovery regarded the dark brown inks used on a Qur'ānic fragment attributed to the 10th century. SEM micrographs disclosed the presence of red blood cells mixed to ink components. The inks were also investigated with Raman spectroscopy. The third case study is the book “Libretto di appunti e memorie del Padre Francesco Zazzera”, dated 17th century that presented a peculiar modification of the black ink that appeared to be faded and turned to a whiteyellowish color. Raman and SEM analyses recognized and documented a biogenic formation of jarosite on top of the ink. It was possible to remove the jarosite layer and to recover the original black ink, thus allowing an easier reading of the text. The fourth and last case analyzed and presented in this paper consists of the measurements on the invaluable Codex Purpureus Rossanensis dated 6th century. Raman spectroscopy here allowed the demonstration of the use of an elderberry lake to obtain a mauve color. This is the first experimental evidence of the use of that particular dye in such an ancient illuminated manuscript. A sort of archeological discovery was also done on this codex with respect to the past misfortunes that occurred to it; by simply observing some traces we were able to support some intriguing hypothesis on its history and vicissitudes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail addresses: [email protected] (M. Bicchieri), fl[email protected], [email protected] (F. Pinzari). 1 Present address: (CREA) Consiglio per la Ricerca in Agricoltura e l'analisi dell'economia Agraria. Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Via della Navicella 2–4, 00184 Roma, Italy.
http://dx.doi.org/10.1016/j.microc.2015.09.028 0026-265X/© 2015 Elsevier B.V. All rights reserved.
The backstage of book or archival document analysis might focus, in the general imagination, on collecting information on the supporting paper or parchment and on the inks, black, white or color used to decorate or write on the support. This is often not the case and, as a matter of fact, the reality of library material analysis often is very different and more articulated and can lead to the occurrence of surprising discoveries.
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This paper presents four case studies focusing on four artifacts of different ages and provenances, all analyzed and studied at the Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario (Icrcpal). The first case deals with two parchment codices that have been found in 2008 during a fishing expedition in the Canale di Sicilia, 60 miles off the coast of Pantelleria (Sicily, Italy). The codices were given at first to the Soprintendenza per i Beni Culturali e Ambientali di Trapani. They have then been handled to the Biblioteca Centrale della Regione Siciliana, where they were left drying without any preliminary washing, while being stored in a suitable box. They were then sent to Icrcpal, for a complete characterization. One codex consists of the remains of about 30 deteriorated parchment sheets (cm 22 × cm 25), with only a few pages still in bifolio (Fig. 1, left). No writing signs or sewing traces are observable to the naked eye under the layer of salt that had covered all the sheets of the volume. The other consists of 40 not assembled parchment sheets (cm 54 × cm 30) and of a particular oversize parchment (cm 320 × cm 54) folded in 6 parts (Fig. 1, right). Also in this case, no graphic signs are observable to the naked eye. The second case deals with one medieval manuscript fragment (Fig. 2) that was found in 2007 buried in the walls of the Great Mosque of Sana'a in Yemen, during restoration works. At that time around 4000 fragments in paper and parchment, dated 7th century–10th century, were found lying hidden in two niches on the western Mosque side, in correspondence to the minaret's wall. The fragments were in very poor condition and a conservation project was established between the Yemeni Antiquities Authority and the Icrcpal, where some samples were analyzed by using non-destructive techniques. The third case study is the booklet “Libretto di appunti e memorie del Padre Francesco Zazzera”, dated 17th century. Francesco Zazzera was a close follower and coworker of St. Filippo Neri, who founded in 1575 a society of secular clergy, called the Congregazione dell'Oratorio. He left numerous writings on the life and the doctrine of the Saint and a booklet containing notes and anecdotes. Such a booklet, consisting of around 40 pages, measures cm 9.3 × cm 13.9 and presented a peculiar modification of the black ink that appeared to be faded and turned to a whiteyellowish color (Fig. 3). The fourth and last case presented in this paper is related with the analyses performed on the invaluable Purple Codex Rossanensis (Fig. 4). The Codex Rossanensis is a 6th century Byzantine illuminated
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Fig. 2. The medieval Yemenite fragment (recto and verso).
manuscript written on purple parchment and conserved at the Museo Diocesano in Rossano Calabro (Cosenza, Italy). The manuscript contains thirteen miniatures hinged on the Life of Christ, one miniature of the four Evangelists, part of the letter to Carpianum arranged in a golden decoration, the illumination of the Apostle Mark inspired by the Sophia; the codex is written in silver and gold on purple parchment with the occasional presence of black inks. In 1917–19 it was restored by Nestore Leoni, a famous miniaturist, whose intervention unfortunately irreversibly modified the aspect of the illuminated pages. Nestore Leoni never documented which materials he used for the restoration. The chemistry laboratory was faced with several questions, namely on the nature of the products applied by Leoni during the restoration, the composition of the pictorial palette used by the miniaturist(s) and the composition of the different inks present in the manuscript. The laboratory also had to provide scientific information that could help in solving a problem of a paramount historical importance: some scholars supposed that the illumination of Mark inspired by the Sophia did not belong to the original manuscript, but had to be dated back to the
Fig. 1. The codices found under the sea. To the left: the first codex (each page cm 22 × cm 25), to the right: the long strip (cm 320 × cm 54) folded in the second codex.
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Fig. 3. The booklet Libretto di appunti e memorie del Padre Francesco Zazzera with faded and no longer readable ink.
12th century. The question was if they were scientific tools that could be used to confirm or reject this hypothesis. Even if no apparent connection can be drawn between the four aforementioned cases, we will show over the development of this paper that there is a common thread amongst them.
2. Materials and methods 2.1. Materials and sampling Direct non-invasive SEM analyses were performed both on the Yemenite fragment and on the 17th century booklet. For biological investigations, small areas (approximately 2 mm2) were collected from damaged regions of the Yemenite sample using watchmaker tweezers or with pressure sensitive tape [1], whereas on the parchment codices and the 17th century booklet, suspected to present a biological contamination, the sampling of the microflora was performed using sterile cellulose acetate membranes (Sartorius, Goettingen, Germany) [2,3]. They are not adhesive, but can retain the spores. Different media were used to determine the microbial charge of aerobic heterotrophic bacteria and fungi: Biolog universal media (BUG, Biolog) for bacteria, MEA (Oxoid) and DG18 (Oxoid) for fungi [4]. The agar plates were kept at 28 °C for 48 h for bacterial growth
while plates with MEA and DG18 media were incubated for fungal growth at 25 °C for 10 days. Model ink samples for the study of the Yemenite ink were necessary. Iron gall ink was prepared by dissolving 0.78 g of powdered oak-gall nuts in 5.0 ml of deionized water for 24 h. After filtration the volume was set to 5.0 ml; 0.31 g of gum arabic and 0.47 g iron (II) sulfate were then added to the solution. For the study of a peculiar pigment found in the Rossanensis, a Sambucus Nigra (pink-violet) lake was prepared from fresh plant fruits that were extracted in a solution of potassium carbonate in distilled water 1:12 w/w. The used ratio between carbonate solution and crushed plant was 10:1 w/w. Two binders for the lake were prepared, one containing yolk and egg white (yolks and egg whites were separated and vigorously whisked, then mixed in the proportion 2:1:1 yolk:egg white:water), the other was a 5% w/V water solution of gum arabic. Each binder was then added to the pulverized lake, until the correct viscosity of the dye was reached. 2.2. Instruments Samples or entire parts were analyzed using a variable pressure (VP) SEM instrument (Zeiss EVO50) fitted with a detector for backscattered electrons (BSE). Chemical characterization of the inorganic constituents of the samples was performed by means of energy dispersion spectroscopy (EDS). Samples analyzed in a high vacuum (HV) mode were previously coated with a gold film 15 nm thick (Baltec Sputter Coater. Gas: argon; working distance: 50 mm; pressure: 0.05 mbar; current: 40 mA; time: 60"). Reference elemental intensities acquired from pure compounds (standards) were used to calibrate SEM–EDS systems [5]. Conventional ZAF correction (correction for Z, atomic, A, absorption and F, fluorescence) was applied. Features of interest were observed with a Leica MZ16 stereoscopic microscope fitted with low temperature fiber optic lights and recorded with a Leica Application Suite digital camera and software (GmbH Wetzlar, Germany). Spectroscopic characterizations were performed with a Renishaw In-via Reflex Raman microscope equipped with a Renishaw diode laser at 785 nm, a 1200 line/mm grating and a Peltier cooled (−70 °C) deep depletion charge-coupled device (CCD RD-VIU, 578 × 384 pixel, with spectral response in the range of 200–1025 nm) optimized for near-infrared and ultraviolet. Nominal spectral resolution was about
Fig. 4. Two pages of the Codex Purpureus Rossanensis. To the left: the people choose between Christ and Barabbas. The miniature contains violet and mauve colors that gave identical Raman spectra, object of our study. To the rights one purple page written in silver. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Photo: Daniele Corciulo, Icrcpal.
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3 cm−1. A Leica DMLM microscope and a color video camera, allow for positioning of the sample. Spectral acquisitions (5–50 accumulations, 50 s each) were performed with a 50× objective (N.A. = 0.75). Under these conditions, the laser spot measures about 20 μm2. The laser power on the sample was 1–0.03 mW, depending on the characteristic of the sample investigated. 3. Results and discussion The four case studies will be discussed separately in the following paragraphs. 3.1. The sea codices Non-destructive Raman measurements were performed on several sheets of the supposed parchment codices, to characterize the nature of the substrate and to study the degradation products deposited on the surfaces. The samples showed differently colored areas, varying from yellow to brown; the latter region could assume different hues from brownreddish to black-brown. The measurements were thoroughly carried out in particular on sheets 13, 14 and 19 of the first codex and on some samples from the second codex and the huge strip. All spectra were evidenced both on the surface and in the bulk, the presence of a cis-isoprenic polymer that completely masked the possible spectrum of the parchment. The isoprenic polymer could be attributed to the penetration of some kind of rubber (the volume could have been protected from humidity using a tissue saturated with a natural rubber) or, most probably, it could have been produced by the action of microorganisms [6]. The presence of diatoms could, in fact, hypothetically explain the finding of these compounds. In fact, some diatoms were found to accumulate polyunsaturated monocyclic sesterand triterpenes, biosynthesized mainly via the mevalonate pathway [7]. In some areas calcite and amorphous carbon were found. In the latter the peaks at lower wavenumbers can be attributed to the formation of metallic oxides, of probable biological origin. Typical spectra are shown in Fig. 5.
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Different species of foraminifera [8] were observed on the surface of all the fragments analyzed. Foraminifera are a large group of aquatic amoeboid protists, which typically produce a shell made of calcium carbonate (CaCO3) [9], as can be observed by the EDX analysis of some shells found on the samples (Fig. 6). Almost nothing of the original asset of parchment could be recognized at SEM, thus confirming the Raman results. Agar-based cultivation methods showed the presence of living fungal spores of few species that can be considered contaminants and dust inhabitants, presumably deposited on the surfaces after their drying. Most of the fragments were found to be covered with an encrusting microbial community covered by a biogenic iron-rich mineral [10,11] containing traces of Mg, Ca and Si (EDX analysis) (Fig. 7). The community formed a biofilm including diatoms, foraminifera micro-sponges and other small biological “objects”. Several studies have documented the formation and occurrence of iron oxides as a result of biotic pathways in natural environments [11,12]. Biotic reactions responsible for the formation of biogenic iron oxides include the microbial oxidation of Fe(II) to Fe(III) by a wide range of microorganisms under both acidic and neutral pH and oxic and anoxic conditions. Iron oxide formation also occurs as a result of passive reactions, where in situ chemical conditions favor the precipitation of the minerals on biological surfaces, namely bacterial cell walls and extracellular material. These reactions are considered passive because the microbes do not gain energy from the oxidation and nucleation processes, but simply act as binding and nucleation surfaces [10]. These iron-rich mineral containing traces of Mg, Ca and Si are typically produced on biological surfaces developing or lying close to marine hydrothermal vents [13]. 3.2. The Yemenite fragment We present only the analyses on one of the fragments received by the Yemenite Authority [14]. The weird discovery regarded the dark brown ink used on the Qur'ānic fragment attributed to the 10th century AD. SEM micrographs disclosed the presence of very peculiar objects (Fig. 8a–b) consisting of flattened spherule-like structures in
Fig. 5. Raman spectra of two areas of one codex found under the sea: top black carbon grain and metallic oxides; bottom spectrum of a cis-isoprenic polymer collected from a brownish area. Excitation λ = 785 nm.
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Fig. 6. To the left: the SEM–QBSD topographical characterization of two foraminifera shells. The compositional contrast between parchment (background) and the shells results from different numbers of backscattered electrons being emitted from areas of the sample differing in atomic number. The backscatter coefficient increases with increasing atomic number and higher atomic number elements (calcium respect to carbon and oxygen) appear brighter in the image. To the right: Elemental Dispersive Spectroscopy (EDS-INCA Energy 250) was used to obtain a chemical characterization of the shells that are mainly composed of calcium.
correspondence of some thick inked signs. These objects were initially attributed to fungal spores or pollen grains embedded in the matrix at the time of the writing or ink manufacturing. A later and more careful analysis of images of the ink's surface by means of scanning electron microscopy introduced the possibility of an unexpected origin of these objects. Comparing the Yemen fragment's images with those reported in literature [15,16] carefully considering profilometry and dimensions (Fig. 8c), the spherules were identified as anucleate erythrocytes mixed to inks' components (Fig. 8c). The occurrence of morphological preservation of anucleate, red blood cells in bloodstains has been reported also in Paleolithic tools, estimated to date back as far as 2 million years, as erythrocytes or red blood cell structures have the ability to keep in shape for centuries [17,18,19]. Repeated measurements of red blood cell diameter were performed on the SEM pictures where elements were entirely visible, using the image analysis software, and the average value obtained for the discoidal elements was 6.3 ± 1.5 μm. All mammalian erythrocytes are biconcave discoidal cells resulting from the loss of organelles and nucleus: flattened and depressed in the center. Erythrocytes are circular, except in the camel family Camelidae, where they are oval. A typical human erythrocyte disk has a diameter of 6–8 μm and a thickness of 2 μm. The red blood cells of other mammals vary between 2.5 and 10 μm. All the other vertebrates possess erythrocytes that keep the nucleus and are thus not biconcave or flattened (i.e., birds, reptiles). Amphibians have the largest red
blood cells (i.e., 16–25 μm in frogs). The cells found in the ink could reasonably be attributed to a mammal since distinguishing mammalian versus non-mammalian (e.g., bird) RBCs morphologically is feasible, and this can be done using SEM. Forensic microscopists attempted to distinguish the species of origin of bloodstains based on RBC diameters. However, this was abandoned due to the very slight or null differences between many species. At the same time the Raman analysis of the black ink provided interesting results: the characteristic Raman peak of iron-gall inks, centered at around 1484 cm−1, appeared shifted at 1491 cm−1. The shift could have been induced by the presence of blood that represents an unusual source of iron. Addition of blood in the manufacturing of iron-tannic inks, without any indication of its amount, is mentioned in ancient middle-eastern manuscripts [20–23], but the scientific literature does not report if its presence could induce modification in the vibrational spectra of inks. It was thus decided to verify this hypothesis, analyzing by Raman spectroscopy some mockups prepared by adding blood to standard laboratory iron-gall ink. The chosen ratio was iron gall/blood 2/1 to obtain a solution with a high content of red-blood cells. For ethical reasons we did not use animal blood, but human provided by two volunteer donors. No spectral differences were detected between the normal iron-gall ink and the “bloody” one (Fig. 9). The position of the four characteristic peaks of iron-gall inks (1348, 1431, 1486 and 1596 cm− 1) did not
Fig. 7. On the left: the SEM–QBSD (low magnification) topographical characterization of the black-rusty crusts that cover the surface of the “pages” of the first codex. The compositional contrast between parchment (background) and the crust results from different numbers of backscattered electrons being emitted from areas of the sample differing in atomic number. On the right: Elemental Dispersive Spectroscopy (EDS-INCA Energy 250) was used to obtain a chemical characterization of the crusts that are mainly composed of Iron, but contains also Mg, Si, and Ca. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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condensed tannins from Mangrove (Rhizophora mangle) and hydrolysable from Chestnut (Castanea sativa), Sumac (Rhus coriaria), Myrobalan (Prunus cerasifera) and Valonea (Quercus macrolepis) have been studied, characterized and compared with those extracted from gall-nuts. From the experimental data it can be inferred that tannins from the same family (i.e., Chestnut and Valonea, Fagacee family) are quite similar, with the main peaks centered around the same wavenumbers. Moreover the possible use of condensed tannins (i.e., Mangrove) is easily recognizable (Table 1) The best correspondence (Table 1) in the characterizing peaks of the original ink and those prepared in laboratory was found with sumac or a plant of the Rhus genus. It is to underline that Raman spectra collected from different vegetal sources of the same genus are quite similar and can present small shifts in the frequency of the peaks. We are now analyzing tannins of Middle Eastern native plants belonging to Rhus genus to find, if possible, the optimal match (Fig. 10). 3.3. The booklet “Libretto di appunti e memorie del Padre Francesco Zazzera”
Fig. 8. Qur'ānic fragment attributed to the 10th century AD; a) stereomicroscopic imaging of the writing; b) SEM micrograph at low magnification (BSD in variable pressure); and c) SEM micrograph in variable pressure showing flattened spherules-like structures in correspondence of some thick inked signs.
change after the addition of blood. The unique difference was recorded in the color of the ink: darker black after blood addition. As a second hypothesis we investigated if the shift of the main peak could depend on the usage of a different source of tannins, other than nutgalls, in its preparation. Results are reported in [24], where
Fig. 9. Raman spectra of: A) standard iron gall ink from Aleppo galls; B) and C) spectra of the iron gall ink after addition of blood (two different donors). Spectra are arbitrary stacked for a better visualization. No shifts in the characteristic peaks of the ink are recorded after blood addition.
The volume III.4, belonging to the Archive of the Congregazione dell'Oratorio in Rome was subjected to spectroscopic analysis to assess its conservation status and investigate the reasons that induced a modification in the color of the ink (from black to yellowish-white) in many pages. The manuscript was analyzed in Raman spectroscopy in different points: unwritten paper, black inks and inks turned to yellowish-white. The analysis of the spectra of the papers showed a severe oxidation of the cellulose support. As regards the ink, the Raman spectra collected from areas free from color variation, indicate the presence of a degraded and perforating iron-gall ink (collapse of the 3 typical bands of the tannin between 1300 and 1700 cm−1), while the yellowish-white areas gave overlapping spectra of iron-gall ink in perfect conditions and jarosite [KFe3 (SO4)2 (OH)6] (Fig. 11), which, as reported in the literature [25], may have been produced by the action of bacteria. The observation under the Raman microscope showed the presence of jarosite crystal deposits on the ink surface. The solubility of the jarosite in water and the concomitant evidence of the excellent state of conservation of the ink below, led us to try the removal of the biogenetic sulfate, by using a swab lightly moistened with water. After performing a removal test under a microscope, Raman spectra were collected. The spectra showed that the removal was almost complete; only in some areas there were still slight traces of jarosite. The method was then applied to all the pages showing the severe degradation of the ink, till the complete removal of the incrustations. In this way the readability of the text was recovered (inset in Fig. 11). The observation under the SEM microscope actually confirmed the presence of jarosite crystal deposits on the ink surface (Fig. 12a). The crystals presented a typical rhombohedral shape (Fig. 12b–d) and appeared associated with a biological biofilm, made of both fungal and bacterial structures (Fig. 12d). Actually a biological origin of jarosite has been widely described, although mainly due to the activity of some chemolithotrophic bacteria, which utilizes ferrous and carbon dioxide as energy and carbon sources in extreme low pH environment such as acid mine drainage areas. In these chemical microenvironments microbial oxidation of Fe2+ ion is often accompanied by the precipitation of Fe3 +-containing minerals such as jarosite, but also goethite and ferrihydrite, which was defined as a biologically induced mineralization [26,27]. Jarosite was found also to nucleate on fungal cell walls [28]. Extracellular polymeric substances (EPS) released by some fungi could serve as nucleation sites for this biomineralization process. The model proposed by Oggerin et al. [28] consists of the creation of Fe3+/Fe2+ rich microdomains in the cell walls of the fungus that induce the supersaturation and precipitation of jarosite. The shift in the proportions of reduced
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Table 1 Position of the characteristic Raman bands of inks produced by using different tannins. Mangrove ink
Chestnut ink
Valonea ink
Myrobalan ink
Iron-gall ink
Sumac ink
1323 1429 1482
1347 1425 1475
1351 1430 1477
1341 1448 1486
1331 1430 1477
1568
1575
1575
1586
1581
1343 1433 1474 1499 1586
a
Frag. 3 ink a a
1491 1587
Attribution ν(C–O) (carboxyilic) Simm ν(COO−); β (C–H); β(O–H) Sciss (CH2); ν(C_C) ring (semicircle stretching); β (C–H) ν(C_C) ring (quadrant stretching)
Broad undefined band with possible sub-structures between 1420 and 1480 cm−1.
and oxidized iron species can be produced by the activity of some bacteria or by electrostatic interaction between soluble ferric iron and the negatively charged groups of the fungal cell wall. The EDS analysis that focused on crystals confirmed the stoichiometry of jarosite (Fig. 12b–c). The microbiological tests gave as result a mixed microflora, not containing species directly related to jarosite formation. Some Penicillium and Aspergillus fungal species, and a few aerobic bacteria grown on the Petri dishes could be related to air contamination. The book underwent a soaking event that left it covered with mud. Possibly the microflora that caused the biogenesis of jarosite in correspondence of inked areas was no longer alive and not culturable at the conditions used in this study. Further investigations are being undertaken to identify microbial and fungal species that caused the precipitation of Fe3+ containing minerals, using a culture independent approach. In our knowledge, this is the first documentation of such kind of degradation on a book, as well as the first restoration treatment attempted in such a condition.
3.4. The Codex purpureus Rossanensis The results of the complete characterization of the codex are reported in [29]. In this paper we will only discuss the discovery of the use of an elderberry lake, never found before in so very ancient manuscripts. The analyses of the palette of the codex Rossanensis, revealed the presence of a mauve color, in which Raman spectrum was not reported in the scientific literature. Its spectral features corresponded to those of an organic compound and the position of the peaks let us suppose that the compound responsible for the color should belong to the family of anthocyanins and related flavonoids. A possible candidate for the mauve color was the elderberry lake, cited in ancient recipe books, but not characterized
Fig. 10. Raman spectra of iron-tannic inks from Mangrove, Myrobalan, Chestnut, Valonea, Sumac, and of iron-gall ink compared with the original ink of the fragment discussed in this paper. Spectra are arbitrary stacked for a better visualization.
from the spectroscopic point of view. It was decided to prepare that lake and to analyze it. It has to be stressed that the spectroscopic direct analysis of dyes and lakes is particularly difficult, both because the dyes used for the lakes are poor Raman scatterers and because they are applied at very low concentrations. Moreover, the colors obtained from natural compounds contain not only the coloring principle, but a set of chemical compounds related to the needs of the plants (glycosides, pectins, proteins, essential oils, vitamins, minerals, carbohydrates) and, concerning elderberry, within each plant source, even belonging to the same family and genus, anthocyanins vary in concentration and chemical structure [30–33]. Usually, when molecular spectroscopies are chosen, the lakes are analyzed by using SERS (Surface Enhanced Raman Spectroscopy), but this involves a direct contact with the original work of art, that is not permitted by the policy of the Italian Ministry for Cultural Heritage. Elderberry, due its usage in medicine and in food, has been thoroughly characterized, in its single components, and spectra from pure or purified compounds — usually dispersed in water solution — are available in literature [34–36], but the direct characterization from a lake applied on a writing support had never been performed before. Fig. 13 reports the Raman spectra of the laboratory elderberry lake compared with a typical spectrum collected from an original mauve area. As can be seen there is a perfect correspondence in the characterizing bands between the two spectra. The enlargement of the bands in the 1200–1600 cm−1 region in the spectrum collected from the original dye is due to the presence of a small amount of carbon black in the original dye, as can be seen in Fig. 13, where the spectrum of carbon black is plotted in the region where the characteristic bands are located. The two intense peaks at 981 and 1009 cm−1 in the elderberry lake are related to the presence of aluminum sulfate, necessary for the manufacturing of the lake.
Fig. 11. Raman spectra of the faded ink the book “Libretto di appunti e memorie del Padre Francesco Zazzera”, before and after removal of the superficial layer of jarosite, which spectrum is reported for comparison. The inset shows on the top the aspect of the faded ink and bottom how it appears after the first jarosite removal attempt. The readability has been recovered. Spectra are arbitrary stacked for a better visualization.
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Fig. 12. Detail of the volume III.4, belonging to the Archive of the Congregazione dell'Oratorio in Rome; a) black inks turned to yellowish-white; b) SEM imaging (BSD in variable pressure) showing the presence of jarosite crystals deposits on the ink surface; crystals presented a typical rhombohedral shape; c) EDS spectra of a crystal; and d) SEM High vacuum imaging on metalized sample: the crystal appeared associated to a biological biofilm, made of both fungal and bacterial structures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A further validation of our attribution was obtained by using FORS (Fiber Optics Reflectance Spectroscopy) at the physics laboratory of Icrcpal. Fig. 14 reports the spectra obtained from the original color and from two elderberry lakes applied on parchment samples by using gum arabic or eggs as binding material. Table 2 reports the position of the peaks. As can be seen, the peaks of the three samples arise at the same wavelength, but the curves obtained with the two binders have a different slope in the 600–800 nm region. In this region the original color presents the same slope of the laboratory sample obtained by mixing the lake with gum arabic. This evidence let us suppose the use of this kind of binder in the Codex. In our knowledge this is the first evidence of the use of an elderberry lake in a very ancient manuscript (6th century). A sort of archeological discovery was also done with respect to the past misfortunes that occurred to the codex, by simply analyzing a few traces (Fig. 15). Diffuse spots affected some of the folios that,
Fig. 13. Raman spectra collected from the original mauve color and the Elderberry (Sambucus nigra) Lake prepared in laboratory. Carbon black spectrum is reported to evidence the enlargement of the peaks in the 1200–1600 cm−1 region, due to the presence of carbon black in the original pigment. Spectra are arbitrary stacked for a better visualization. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
when observed at the stereomicroscope in transmitted light, appeared translucent because of the parchment erosion in those areas. Our hypothesis is that, at a certain point during the last century, the Codex Rossanensis underwent a bacterial attack that caused severe damage to a binding that is now missed, and to the initial pages, resulting in the spotted alteration of the codex. The putative bacterial attack degraded the collagen fibers, leaving the parchment perforated and weak, thus requiring the intervention of restorers. After the intervention, based on gelatin application on the first illuminated folios to strengthen them, the degraded areas showed with a glassy appearance. The bacterial attack to the Codex Rossanensis is only a hypothesis because it could not be confirmed analytically (no samples could be taken for SEM analysis, and the size of the manuscript prevented it to be inserted in the SEM chamber), but it is based on comparison with several case studies well documented by Piñar and other specialists in this research field [37–39]. Evidence that strengthen the hypothesis of a bacterial attack to the codex is observable where the purple stains are scattered close to the text written in silver ink. In these areas the stains “avoid” the writing because of copper impurities present in the silver ink that are toxic to the
Fig. 14. FORS spectra of the original mauve lake and two elderberry lakes dispersed in gum arabic or in egg. The inset shows the logarithmic transformation of reflectance for the original dye and the elderberry lake in gum arabic in characterizing 450–650 nm region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Peaks position (nm) in FORS spectra for the original pink-mauve lake and the elderberry lake dispersed in two different binders (gum arabic and egg). Original mauve color peaks (nm)
Laboratory elderberry lake—gum arabic peaks (nm)
Laboratory elderberry lake—egg peaks (nm)
λ1 λ2 λ3
λ1 λ2 λ3
λ1 λ2 λ3
496.0 ± 1.0 524.1 ± 0.8 564.0 ± 0.2
496.0 ± 1.0 525.1 ± 0.7 564.2 ± 0.3
496.0 ± 0.9 525.1 ± 0.8 564.2 ± 0.2
bacteria. The same phenomenon has been documented in other case studies [37–39]. These bacteria (phylum Actinobacteria) play a major role in the deterioration of ancient parchments. They are filamentous halophilic bacteria known to produce collagenases that are capable of destroying collagen by their hydrolytic activity. They are alkaliphiles and inclined to develop on parchment that, during its manufacture, is processed with alkaline materials such as lime and chalk [38,39].
4. Conclusions The link between the case studies presented in this paper is the discovery, in objects of historical interest, of products or compounds that
Fig. 15. The Codex Rossanensis: a) transmitted light image of p. 15 showing scattered translucent areas where the parchment was thinned by the bacteria; b) detail in natural light of the area of p. 15 showing a direct correlation between the purple stains and the thinned areas (image obtained with a stereomicroscope Leica M16); and c) the typical damage caused by bacteria of the phylum Actinobacteria on a 16th century parchment (showed as comparison respect to the codex). d) Detail in natural light of silver writing on p. 17 showing a peculiar pattern of the purple stains from bacteria that do not overlap onto the ink (image obtained with a stereomicroscope Leica M16). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
are not related with the original materials, but with the vicissitudes of the original documents or their peculiar manufacture. Furthermore, the comparative studies carried out by the laboratories of chemistry and biology highlight the need of a close collaboration between the different competences: many chemical modifications in the original structures can be explained only as a result of biological changes and the latter can be induced by particular chemical compounds present within the documents or in the external environment. To conclude we can say with a smile, that in the analysis of library materials we “have seen things you people wouldn't believe…” [40]. Acknowledgment We would like to thank the colleagues Lorena Botti, Daniele Ruggiero and Maria Teresa Tanasi of the Icrcpal physics laboratory for their collaboration in the FORS characterization of the lakes prepared in the chemistry laboratory and for sharing their FORS results on the original lake. References [1] F. Pinzari, M. Montanari, A. Michaelsen, G. Piñar, Analytical protocols for the assessment of biological damage in historical documents, Coalition 19 (2010) 6–13. [2] F. Cappitelli, G. Pasquariello, G. Tarsitani, C. Sorlini, Scripta manent? Assessing microbial risk to paper heritage, Trends Microbiol. 18 (12) (2010) 538–542. [3] P. Principi, F. Villa, C. Sorlini, F. Cappitelli, Molecular studies of microbial community structure on stained pages of Leonardo da Vinci's Atlantic Codex, Microb. Ecol. 61 (2011) 214–222. [4] F. Pinzari, P. Colaizzi, O. Maggi, A.M. Persiani, R. Schütz, I. Rabin, Fungal bioleaching of mineral components in a twentieth-century illuminated parchment, Anal. Bioanal. Chem. 402 (4) (2012) 1541–1550. [5] J. Goldstein, D. Newbury, D. Joy, C. Lyman, P. Echlin, E. Lifshin, et al., Scanning Electron Microscopy and X-ray Microanalysis, third ed. Springer Science, Business Media Inc., New York, 2003. [6] E. Swiezewska, W. Danikiewicz, Review: polyisoprenoids: structure, biosynthesis and function, Prog. Lipid Res. 44 (2005) 235–258. [7] G. Massé, S.T. Belt, S.J. Rowland, Biosynthesis of unusual monocyclic alkenes by the diatom Rhizosolenia setigera (Brightwell), Phytochemistry 65 (2004) 1101–1106. [8] B.K. Sen Gupta (Ed.), Modern Foraminifera, Springer, Netherlands, 1983. [9] C. Hemleben, M. Spindler, O.R. Anderson, Modern Planktonic Foraminifera, Springer, New York, 1989. [10] D. Fortin, S. Langley, Formation and occurrence of biogenic iron-rich minerals, EarthSci. Rev. 72 (2005) 1–19. [11] D. Fortin, F.G. Ferris, S.D. Scott, Formation of Fe-silicates and Fe-oxides on bacterial surfaces in hydrothermal deposits collected near the Southern Explorer Ridge in the Northeast Pacific Ocean, Am. Mineral. 83 (1998) 1399–1408. [12] J.F. Banfield, S.A. Welch, H. Zhang, T.T. Ebert, L.P. Penn, Aggregation-based crystal growth and microstructure development in natural iron oxyhydroxide biomineralization products, Science 289 (2000) 751–754. [13] Dando, et al., Hydrothermalism in the Mediterranean Sea, Prog. Oceanogr. 44 (1999) 333–367. [14] A. Batori, C. Casetti Brach, M. Bicchieri, M. Di Bella, F. Pinzari, Yemen: i frammenti ritrovati nella Grande Moschea di Sana'a, Restauro. , Edizioni MP Mirabilia srl, Roma, 2009 16–17. [15] T.H. Loy, Prehistoric blood residues: detection on tool surfaces and identification of species of origin, Science 220 (1983) 1269–1271. [16] P. Hortolà, SEM characterization of blood stains on stone tools, Microscope 40 (2) (1992) 111–113. [17] P. Hortolà, Secondary-electron SEM bioimaging of human erythrocytes in bloodstains on high-carbon steel substrate without specimen preparation, Micron 39 (2008) 53–55. [18] P. Hortolà, Application of SEM to the study of red blood cells in forensic bloodstains. Microscopy and Analysis 40 (1994) 19–21 (UK) and 28 (1994) 21–23 (EU). [19] C.M. Hawkey, P.M. Bennett, S.C. Gascoyne, M.G. Hart, J.K. Kirkwood, Erythrocyte size, number and haemoglobin content in vertebrates, Br. J. Haematol. 77 (1991) 392–397. [20] M. Zaki, Early Arabic bookmaking techniques as described by al-Rāzī in his recently rediscovered Zīnat al-Katabah, J. Islam. Manuscr. 2 (2011) 223–234. [21] al-Qalalūsī (al-Andalusī), Tuḥaf al-khawāṣṣ fī ṭuraf al-khawāṣṣ: fī ṣanʻat al-amiddah wa-al-aṣbāgh wa-al-adʹhān Ḥusām Aḥmad Muḫtār al-ʻAbādī, al-Iskandariyya, 2007. [22] M. Levey, Some black inks in early Medieval Jewish Literature, Chymia 9 (1964) 27–31. [23] A. Schopen, Tinten und Tuschen des arabisch-islamischen Mittelalters: Dokumentation, Analyse, Rekonstruktion: ein Beitrag zur materiellen Kultur des Vorderen Orients, Vandenhoeck & Ruprecht, Gottingen, 2006. [24] M. Bicchieri, M. Monti, G. Piantanida, A. Sodo, Non-destructive spectroscopic investigation on historic Yemenite scriptorial fragments: evidence of different degradation and recipes for iron tannic inks, Anal. Bioanal. Chem. 405 (8) (2013) 2713–2721. [25] G.A. Desborough, et al., Mineralogical and chemical characteristics of some natural jarosites, Geochim. Cosmochim. Acta 74 (2010) 1041–1056.
M. Bicchieri, F. Pinzari / Microchemical Journal 124 (2016) 568–577 [26] K. Sasaki, H. Konno, Morphology of jarosite-group compounds precipitated from biologically and chemically oxidized Fe ions, Can. Mineral. 38 (1) (2000) 45–56. [27] H.L. Dong, Mineral–microbe interactions: a review, Front. Earth Sci. China 4 (2) (2010) 127–147. [28] M. Oggerin, F. Tornos, N. Rodríguez, C. Del Moral, M. Sánchez-Roman, R. Amils, Biomineralization of jarosite by Purpureocillium lilacinum, an acidophilic fungi isolated from Rio Tinto, Goldschmidt 2013 Conference Abstracts 2013, p. 1880, http://dx. doi.org/10.1180/minmag.2013.077.5.15 (www.minersoc.org). [29] M. Bicchieri, The purple Codex Rossanensis: spectroscopic characterization and first evidence of the use of the Elderberry Lake in a 6th century manuscript, Environ. Sci. Pollut. Res. 21 (24) (2014) 14146–14157. [30] S.E. Gebhardt, J.M. Harnly, S.A. Bhagwat, G.R. Beecher, et al., USDA's flavonoid database: flavonoids in fruit, http://www.villagewineryandvineyards.com/USDA-elderberry-Flavonoid-chart.pdf2002. [31] L. Jungmin, E.F. Chad, Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European elderberry (S. nigra) cultivars, J. Sci. Food Agric. 87 (2007) 2665–2675. [32] M. Buchweitz, G. Gudi, R. Carle, R.C. Kammer, R. Dietmar, H. Schulz, Systematic investigations of anthocyanin–metal interactions by Raman spectroscopy, J. Raman Spectrosc. 43 (12) (2012) 2001–2007. [33] European Medicines Agency Assessment report on Sambucus nigra L., fructus, http:// www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_HMPC_assessment_ report/2013/04/WC500142245.pdf2013.
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Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Discoveries and oddities in library materials Marina Bicchieri a,⁎, Flavia Pinzari b,c,1 a b c
Laboratorio di Chimica, Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Via Milano 76, 00184 Roma, Italy Laboratorio di Biologia, Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Via Milano 76, 00184 Roma, Italy Natural History Museum of London, Department of Life Sciences, Cromwell Rd, London SW7 5BD, United Kingdom
a r t i c l e
i n f o
Article history: Received 14 July 2015 Received in revised form 28 September 2015 Accepted 28 September 2015 Available online 9 October 2015 Keywords: Raman SEM–EDS Inks Jarosite Elderberry
a b s t r a c t Surprising discoveries can happen while analyzing library materials. This paper presents four case studies focusing on four artifacts of different ages and provenances, all analyzed and studied at the Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario (Icrcpal). The common denominator amongst the case studies presented in this work, lies in the need for a strong interconnection between the analysis of biological issues and chemical data. The interplay between the two complementary approaches is paramount for the correct interpretation of the status of the material under examination as, as will be shown in the following case studies, chemical data could find an interpretation only in the face of biological observations and vice versa. The first case we here present, deals with two parchment codices that have been found in 2008 during a fishing expedition in the Canale di Sicilia. At that time a deep-sea fishing boat belonging to the fleet of Mazara del Vallo caught in its nets, two artifacts, leading to the first discovery of library materials in the deep sea. The Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS) analyses allowed for the creation of a compositional map of the surface of the samples, where several microscopic encrusting sea organisms and biogenic materials were also observed. Almost nothing of the original structure of the parchment (of which the object is presumably made) could be recognized at SEM. The absence of the typical collagen features was confirmed by Raman analyses that could only collect spectra of an isoprenic-type polymer of putative biogenic origin. The second case deals with fragments of medieval manuscripts that were found in 2007 buried in the walls of the Great Mosque of Sana'a in Yemen. An outstanding discovery regarded the dark brown inks used on a Qur'ānic fragment attributed to the 10th century. SEM micrographs disclosed the presence of red blood cells mixed to ink components. The inks were also investigated with Raman spectroscopy. The third case study is the book “Libretto di appunti e memorie del Padre Francesco Zazzera”, dated 17th century that presented a peculiar modification of the black ink that appeared to be faded and turned to a whiteyellowish color. Raman and SEM analyses recognized and documented a biogenic formation of jarosite on top of the ink. It was possible to remove the jarosite layer and to recover the original black ink, thus allowing an easier reading of the text. The fourth and last case analyzed and presented in this paper consists of the measurements on the invaluable Codex Purpureus Rossanensis dated 6th century. Raman spectroscopy here allowed the demonstration of the use of an elderberry lake to obtain a mauve color. This is the first experimental evidence of the use of that particular dye in such an ancient illuminated manuscript. A sort of archeological discovery was also done on this codex with respect to the past misfortunes that occurred to it; by simply observing some traces we were able to support some intriguing hypothesis on its history and vicissitudes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail addresses: [email protected] (M. Bicchieri), fl[email protected], [email protected] (F. Pinzari). 1 Present address: (CREA) Consiglio per la Ricerca in Agricoltura e l'analisi dell'economia Agraria. Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Via della Navicella 2–4, 00184 Roma, Italy.
http://dx.doi.org/10.1016/j.microc.2015.09.028 0026-265X/© 2015 Elsevier B.V. All rights reserved.
The backstage of book or archival document analysis might focus, in the general imagination, on collecting information on the supporting paper or parchment and on the inks, black, white or color used to decorate or write on the support. This is often not the case and, as a matter of fact, the reality of library material analysis often is very different and more articulated and can lead to the occurrence of surprising discoveries.
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This paper presents four case studies focusing on four artifacts of different ages and provenances, all analyzed and studied at the Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario (Icrcpal). The first case deals with two parchment codices that have been found in 2008 during a fishing expedition in the Canale di Sicilia, 60 miles off the coast of Pantelleria (Sicily, Italy). The codices were given at first to the Soprintendenza per i Beni Culturali e Ambientali di Trapani. They have then been handled to the Biblioteca Centrale della Regione Siciliana, where they were left drying without any preliminary washing, while being stored in a suitable box. They were then sent to Icrcpal, for a complete characterization. One codex consists of the remains of about 30 deteriorated parchment sheets (cm 22 × cm 25), with only a few pages still in bifolio (Fig. 1, left). No writing signs or sewing traces are observable to the naked eye under the layer of salt that had covered all the sheets of the volume. The other consists of 40 not assembled parchment sheets (cm 54 × cm 30) and of a particular oversize parchment (cm 320 × cm 54) folded in 6 parts (Fig. 1, right). Also in this case, no graphic signs are observable to the naked eye. The second case deals with one medieval manuscript fragment (Fig. 2) that was found in 2007 buried in the walls of the Great Mosque of Sana'a in Yemen, during restoration works. At that time around 4000 fragments in paper and parchment, dated 7th century–10th century, were found lying hidden in two niches on the western Mosque side, in correspondence to the minaret's wall. The fragments were in very poor condition and a conservation project was established between the Yemeni Antiquities Authority and the Icrcpal, where some samples were analyzed by using non-destructive techniques. The third case study is the booklet “Libretto di appunti e memorie del Padre Francesco Zazzera”, dated 17th century. Francesco Zazzera was a close follower and coworker of St. Filippo Neri, who founded in 1575 a society of secular clergy, called the Congregazione dell'Oratorio. He left numerous writings on the life and the doctrine of the Saint and a booklet containing notes and anecdotes. Such a booklet, consisting of around 40 pages, measures cm 9.3 × cm 13.9 and presented a peculiar modification of the black ink that appeared to be faded and turned to a whiteyellowish color (Fig. 3). The fourth and last case presented in this paper is related with the analyses performed on the invaluable Purple Codex Rossanensis (Fig. 4). The Codex Rossanensis is a 6th century Byzantine illuminated
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Fig. 2. The medieval Yemenite fragment (recto and verso).
manuscript written on purple parchment and conserved at the Museo Diocesano in Rossano Calabro (Cosenza, Italy). The manuscript contains thirteen miniatures hinged on the Life of Christ, one miniature of the four Evangelists, part of the letter to Carpianum arranged in a golden decoration, the illumination of the Apostle Mark inspired by the Sophia; the codex is written in silver and gold on purple parchment with the occasional presence of black inks. In 1917–19 it was restored by Nestore Leoni, a famous miniaturist, whose intervention unfortunately irreversibly modified the aspect of the illuminated pages. Nestore Leoni never documented which materials he used for the restoration. The chemistry laboratory was faced with several questions, namely on the nature of the products applied by Leoni during the restoration, the composition of the pictorial palette used by the miniaturist(s) and the composition of the different inks present in the manuscript. The laboratory also had to provide scientific information that could help in solving a problem of a paramount historical importance: some scholars supposed that the illumination of Mark inspired by the Sophia did not belong to the original manuscript, but had to be dated back to the
Fig. 1. The codices found under the sea. To the left: the first codex (each page cm 22 × cm 25), to the right: the long strip (cm 320 × cm 54) folded in the second codex.
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Fig. 3. The booklet Libretto di appunti e memorie del Padre Francesco Zazzera with faded and no longer readable ink.
12th century. The question was if they were scientific tools that could be used to confirm or reject this hypothesis. Even if no apparent connection can be drawn between the four aforementioned cases, we will show over the development of this paper that there is a common thread amongst them.
2. Materials and methods 2.1. Materials and sampling Direct non-invasive SEM analyses were performed both on the Yemenite fragment and on the 17th century booklet. For biological investigations, small areas (approximately 2 mm2) were collected from damaged regions of the Yemenite sample using watchmaker tweezers or with pressure sensitive tape [1], whereas on the parchment codices and the 17th century booklet, suspected to present a biological contamination, the sampling of the microflora was performed using sterile cellulose acetate membranes (Sartorius, Goettingen, Germany) [2,3]. They are not adhesive, but can retain the spores. Different media were used to determine the microbial charge of aerobic heterotrophic bacteria and fungi: Biolog universal media (BUG, Biolog) for bacteria, MEA (Oxoid) and DG18 (Oxoid) for fungi [4]. The agar plates were kept at 28 °C for 48 h for bacterial growth
while plates with MEA and DG18 media were incubated for fungal growth at 25 °C for 10 days. Model ink samples for the study of the Yemenite ink were necessary. Iron gall ink was prepared by dissolving 0.78 g of powdered oak-gall nuts in 5.0 ml of deionized water for 24 h. After filtration the volume was set to 5.0 ml; 0.31 g of gum arabic and 0.47 g iron (II) sulfate were then added to the solution. For the study of a peculiar pigment found in the Rossanensis, a Sambucus Nigra (pink-violet) lake was prepared from fresh plant fruits that were extracted in a solution of potassium carbonate in distilled water 1:12 w/w. The used ratio between carbonate solution and crushed plant was 10:1 w/w. Two binders for the lake were prepared, one containing yolk and egg white (yolks and egg whites were separated and vigorously whisked, then mixed in the proportion 2:1:1 yolk:egg white:water), the other was a 5% w/V water solution of gum arabic. Each binder was then added to the pulverized lake, until the correct viscosity of the dye was reached. 2.2. Instruments Samples or entire parts were analyzed using a variable pressure (VP) SEM instrument (Zeiss EVO50) fitted with a detector for backscattered electrons (BSE). Chemical characterization of the inorganic constituents of the samples was performed by means of energy dispersion spectroscopy (EDS). Samples analyzed in a high vacuum (HV) mode were previously coated with a gold film 15 nm thick (Baltec Sputter Coater. Gas: argon; working distance: 50 mm; pressure: 0.05 mbar; current: 40 mA; time: 60"). Reference elemental intensities acquired from pure compounds (standards) were used to calibrate SEM–EDS systems [5]. Conventional ZAF correction (correction for Z, atomic, A, absorption and F, fluorescence) was applied. Features of interest were observed with a Leica MZ16 stereoscopic microscope fitted with low temperature fiber optic lights and recorded with a Leica Application Suite digital camera and software (GmbH Wetzlar, Germany). Spectroscopic characterizations were performed with a Renishaw In-via Reflex Raman microscope equipped with a Renishaw diode laser at 785 nm, a 1200 line/mm grating and a Peltier cooled (−70 °C) deep depletion charge-coupled device (CCD RD-VIU, 578 × 384 pixel, with spectral response in the range of 200–1025 nm) optimized for near-infrared and ultraviolet. Nominal spectral resolution was about
Fig. 4. Two pages of the Codex Purpureus Rossanensis. To the left: the people choose between Christ and Barabbas. The miniature contains violet and mauve colors that gave identical Raman spectra, object of our study. To the rights one purple page written in silver. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Photo: Daniele Corciulo, Icrcpal.
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3 cm−1. A Leica DMLM microscope and a color video camera, allow for positioning of the sample. Spectral acquisitions (5–50 accumulations, 50 s each) were performed with a 50× objective (N.A. = 0.75). Under these conditions, the laser spot measures about 20 μm2. The laser power on the sample was 1–0.03 mW, depending on the characteristic of the sample investigated. 3. Results and discussion The four case studies will be discussed separately in the following paragraphs. 3.1. The sea codices Non-destructive Raman measurements were performed on several sheets of the supposed parchment codices, to characterize the nature of the substrate and to study the degradation products deposited on the surfaces. The samples showed differently colored areas, varying from yellow to brown; the latter region could assume different hues from brownreddish to black-brown. The measurements were thoroughly carried out in particular on sheets 13, 14 and 19 of the first codex and on some samples from the second codex and the huge strip. All spectra were evidenced both on the surface and in the bulk, the presence of a cis-isoprenic polymer that completely masked the possible spectrum of the parchment. The isoprenic polymer could be attributed to the penetration of some kind of rubber (the volume could have been protected from humidity using a tissue saturated with a natural rubber) or, most probably, it could have been produced by the action of microorganisms [6]. The presence of diatoms could, in fact, hypothetically explain the finding of these compounds. In fact, some diatoms were found to accumulate polyunsaturated monocyclic sesterand triterpenes, biosynthesized mainly via the mevalonate pathway [7]. In some areas calcite and amorphous carbon were found. In the latter the peaks at lower wavenumbers can be attributed to the formation of metallic oxides, of probable biological origin. Typical spectra are shown in Fig. 5.
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Different species of foraminifera [8] were observed on the surface of all the fragments analyzed. Foraminifera are a large group of aquatic amoeboid protists, which typically produce a shell made of calcium carbonate (CaCO3) [9], as can be observed by the EDX analysis of some shells found on the samples (Fig. 6). Almost nothing of the original asset of parchment could be recognized at SEM, thus confirming the Raman results. Agar-based cultivation methods showed the presence of living fungal spores of few species that can be considered contaminants and dust inhabitants, presumably deposited on the surfaces after their drying. Most of the fragments were found to be covered with an encrusting microbial community covered by a biogenic iron-rich mineral [10,11] containing traces of Mg, Ca and Si (EDX analysis) (Fig. 7). The community formed a biofilm including diatoms, foraminifera micro-sponges and other small biological “objects”. Several studies have documented the formation and occurrence of iron oxides as a result of biotic pathways in natural environments [11,12]. Biotic reactions responsible for the formation of biogenic iron oxides include the microbial oxidation of Fe(II) to Fe(III) by a wide range of microorganisms under both acidic and neutral pH and oxic and anoxic conditions. Iron oxide formation also occurs as a result of passive reactions, where in situ chemical conditions favor the precipitation of the minerals on biological surfaces, namely bacterial cell walls and extracellular material. These reactions are considered passive because the microbes do not gain energy from the oxidation and nucleation processes, but simply act as binding and nucleation surfaces [10]. These iron-rich mineral containing traces of Mg, Ca and Si are typically produced on biological surfaces developing or lying close to marine hydrothermal vents [13]. 3.2. The Yemenite fragment We present only the analyses on one of the fragments received by the Yemenite Authority [14]. The weird discovery regarded the dark brown ink used on the Qur'ānic fragment attributed to the 10th century AD. SEM micrographs disclosed the presence of very peculiar objects (Fig. 8a–b) consisting of flattened spherule-like structures in
Fig. 5. Raman spectra of two areas of one codex found under the sea: top black carbon grain and metallic oxides; bottom spectrum of a cis-isoprenic polymer collected from a brownish area. Excitation λ = 785 nm.
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Fig. 6. To the left: the SEM–QBSD topographical characterization of two foraminifera shells. The compositional contrast between parchment (background) and the shells results from different numbers of backscattered electrons being emitted from areas of the sample differing in atomic number. The backscatter coefficient increases with increasing atomic number and higher atomic number elements (calcium respect to carbon and oxygen) appear brighter in the image. To the right: Elemental Dispersive Spectroscopy (EDS-INCA Energy 250) was used to obtain a chemical characterization of the shells that are mainly composed of calcium.
correspondence of some thick inked signs. These objects were initially attributed to fungal spores or pollen grains embedded in the matrix at the time of the writing or ink manufacturing. A later and more careful analysis of images of the ink's surface by means of scanning electron microscopy introduced the possibility of an unexpected origin of these objects. Comparing the Yemen fragment's images with those reported in literature [15,16] carefully considering profilometry and dimensions (Fig. 8c), the spherules were identified as anucleate erythrocytes mixed to inks' components (Fig. 8c). The occurrence of morphological preservation of anucleate, red blood cells in bloodstains has been reported also in Paleolithic tools, estimated to date back as far as 2 million years, as erythrocytes or red blood cell structures have the ability to keep in shape for centuries [17,18,19]. Repeated measurements of red blood cell diameter were performed on the SEM pictures where elements were entirely visible, using the image analysis software, and the average value obtained for the discoidal elements was 6.3 ± 1.5 μm. All mammalian erythrocytes are biconcave discoidal cells resulting from the loss of organelles and nucleus: flattened and depressed in the center. Erythrocytes are circular, except in the camel family Camelidae, where they are oval. A typical human erythrocyte disk has a diameter of 6–8 μm and a thickness of 2 μm. The red blood cells of other mammals vary between 2.5 and 10 μm. All the other vertebrates possess erythrocytes that keep the nucleus and are thus not biconcave or flattened (i.e., birds, reptiles). Amphibians have the largest red
blood cells (i.e., 16–25 μm in frogs). The cells found in the ink could reasonably be attributed to a mammal since distinguishing mammalian versus non-mammalian (e.g., bird) RBCs morphologically is feasible, and this can be done using SEM. Forensic microscopists attempted to distinguish the species of origin of bloodstains based on RBC diameters. However, this was abandoned due to the very slight or null differences between many species. At the same time the Raman analysis of the black ink provided interesting results: the characteristic Raman peak of iron-gall inks, centered at around 1484 cm−1, appeared shifted at 1491 cm−1. The shift could have been induced by the presence of blood that represents an unusual source of iron. Addition of blood in the manufacturing of iron-tannic inks, without any indication of its amount, is mentioned in ancient middle-eastern manuscripts [20–23], but the scientific literature does not report if its presence could induce modification in the vibrational spectra of inks. It was thus decided to verify this hypothesis, analyzing by Raman spectroscopy some mockups prepared by adding blood to standard laboratory iron-gall ink. The chosen ratio was iron gall/blood 2/1 to obtain a solution with a high content of red-blood cells. For ethical reasons we did not use animal blood, but human provided by two volunteer donors. No spectral differences were detected between the normal iron-gall ink and the “bloody” one (Fig. 9). The position of the four characteristic peaks of iron-gall inks (1348, 1431, 1486 and 1596 cm− 1) did not
Fig. 7. On the left: the SEM–QBSD (low magnification) topographical characterization of the black-rusty crusts that cover the surface of the “pages” of the first codex. The compositional contrast between parchment (background) and the crust results from different numbers of backscattered electrons being emitted from areas of the sample differing in atomic number. On the right: Elemental Dispersive Spectroscopy (EDS-INCA Energy 250) was used to obtain a chemical characterization of the crusts that are mainly composed of Iron, but contains also Mg, Si, and Ca. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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condensed tannins from Mangrove (Rhizophora mangle) and hydrolysable from Chestnut (Castanea sativa), Sumac (Rhus coriaria), Myrobalan (Prunus cerasifera) and Valonea (Quercus macrolepis) have been studied, characterized and compared with those extracted from gall-nuts. From the experimental data it can be inferred that tannins from the same family (i.e., Chestnut and Valonea, Fagacee family) are quite similar, with the main peaks centered around the same wavenumbers. Moreover the possible use of condensed tannins (i.e., Mangrove) is easily recognizable (Table 1) The best correspondence (Table 1) in the characterizing peaks of the original ink and those prepared in laboratory was found with sumac or a plant of the Rhus genus. It is to underline that Raman spectra collected from different vegetal sources of the same genus are quite similar and can present small shifts in the frequency of the peaks. We are now analyzing tannins of Middle Eastern native plants belonging to Rhus genus to find, if possible, the optimal match (Fig. 10). 3.3. The booklet “Libretto di appunti e memorie del Padre Francesco Zazzera”
Fig. 8. Qur'ānic fragment attributed to the 10th century AD; a) stereomicroscopic imaging of the writing; b) SEM micrograph at low magnification (BSD in variable pressure); and c) SEM micrograph in variable pressure showing flattened spherules-like structures in correspondence of some thick inked signs.
change after the addition of blood. The unique difference was recorded in the color of the ink: darker black after blood addition. As a second hypothesis we investigated if the shift of the main peak could depend on the usage of a different source of tannins, other than nutgalls, in its preparation. Results are reported in [24], where
Fig. 9. Raman spectra of: A) standard iron gall ink from Aleppo galls; B) and C) spectra of the iron gall ink after addition of blood (two different donors). Spectra are arbitrary stacked for a better visualization. No shifts in the characteristic peaks of the ink are recorded after blood addition.
The volume III.4, belonging to the Archive of the Congregazione dell'Oratorio in Rome was subjected to spectroscopic analysis to assess its conservation status and investigate the reasons that induced a modification in the color of the ink (from black to yellowish-white) in many pages. The manuscript was analyzed in Raman spectroscopy in different points: unwritten paper, black inks and inks turned to yellowish-white. The analysis of the spectra of the papers showed a severe oxidation of the cellulose support. As regards the ink, the Raman spectra collected from areas free from color variation, indicate the presence of a degraded and perforating iron-gall ink (collapse of the 3 typical bands of the tannin between 1300 and 1700 cm−1), while the yellowish-white areas gave overlapping spectra of iron-gall ink in perfect conditions and jarosite [KFe3 (SO4)2 (OH)6] (Fig. 11), which, as reported in the literature [25], may have been produced by the action of bacteria. The observation under the Raman microscope showed the presence of jarosite crystal deposits on the ink surface. The solubility of the jarosite in water and the concomitant evidence of the excellent state of conservation of the ink below, led us to try the removal of the biogenetic sulfate, by using a swab lightly moistened with water. After performing a removal test under a microscope, Raman spectra were collected. The spectra showed that the removal was almost complete; only in some areas there were still slight traces of jarosite. The method was then applied to all the pages showing the severe degradation of the ink, till the complete removal of the incrustations. In this way the readability of the text was recovered (inset in Fig. 11). The observation under the SEM microscope actually confirmed the presence of jarosite crystal deposits on the ink surface (Fig. 12a). The crystals presented a typical rhombohedral shape (Fig. 12b–d) and appeared associated with a biological biofilm, made of both fungal and bacterial structures (Fig. 12d). Actually a biological origin of jarosite has been widely described, although mainly due to the activity of some chemolithotrophic bacteria, which utilizes ferrous and carbon dioxide as energy and carbon sources in extreme low pH environment such as acid mine drainage areas. In these chemical microenvironments microbial oxidation of Fe2+ ion is often accompanied by the precipitation of Fe3 +-containing minerals such as jarosite, but also goethite and ferrihydrite, which was defined as a biologically induced mineralization [26,27]. Jarosite was found also to nucleate on fungal cell walls [28]. Extracellular polymeric substances (EPS) released by some fungi could serve as nucleation sites for this biomineralization process. The model proposed by Oggerin et al. [28] consists of the creation of Fe3+/Fe2+ rich microdomains in the cell walls of the fungus that induce the supersaturation and precipitation of jarosite. The shift in the proportions of reduced
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Table 1 Position of the characteristic Raman bands of inks produced by using different tannins. Mangrove ink
Chestnut ink
Valonea ink
Myrobalan ink
Iron-gall ink
Sumac ink
1323 1429 1482
1347 1425 1475
1351 1430 1477
1341 1448 1486
1331 1430 1477
1568
1575
1575
1586
1581
1343 1433 1474 1499 1586
a
Frag. 3 ink a a
1491 1587
Attribution ν(C–O) (carboxyilic) Simm ν(COO−); β (C–H); β(O–H) Sciss (CH2); ν(C_C) ring (semicircle stretching); β (C–H) ν(C_C) ring (quadrant stretching)
Broad undefined band with possible sub-structures between 1420 and 1480 cm−1.
and oxidized iron species can be produced by the activity of some bacteria or by electrostatic interaction between soluble ferric iron and the negatively charged groups of the fungal cell wall. The EDS analysis that focused on crystals confirmed the stoichiometry of jarosite (Fig. 12b–c). The microbiological tests gave as result a mixed microflora, not containing species directly related to jarosite formation. Some Penicillium and Aspergillus fungal species, and a few aerobic bacteria grown on the Petri dishes could be related to air contamination. The book underwent a soaking event that left it covered with mud. Possibly the microflora that caused the biogenesis of jarosite in correspondence of inked areas was no longer alive and not culturable at the conditions used in this study. Further investigations are being undertaken to identify microbial and fungal species that caused the precipitation of Fe3+ containing minerals, using a culture independent approach. In our knowledge, this is the first documentation of such kind of degradation on a book, as well as the first restoration treatment attempted in such a condition.
3.4. The Codex purpureus Rossanensis The results of the complete characterization of the codex are reported in [29]. In this paper we will only discuss the discovery of the use of an elderberry lake, never found before in so very ancient manuscripts. The analyses of the palette of the codex Rossanensis, revealed the presence of a mauve color, in which Raman spectrum was not reported in the scientific literature. Its spectral features corresponded to those of an organic compound and the position of the peaks let us suppose that the compound responsible for the color should belong to the family of anthocyanins and related flavonoids. A possible candidate for the mauve color was the elderberry lake, cited in ancient recipe books, but not characterized
Fig. 10. Raman spectra of iron-tannic inks from Mangrove, Myrobalan, Chestnut, Valonea, Sumac, and of iron-gall ink compared with the original ink of the fragment discussed in this paper. Spectra are arbitrary stacked for a better visualization.
from the spectroscopic point of view. It was decided to prepare that lake and to analyze it. It has to be stressed that the spectroscopic direct analysis of dyes and lakes is particularly difficult, both because the dyes used for the lakes are poor Raman scatterers and because they are applied at very low concentrations. Moreover, the colors obtained from natural compounds contain not only the coloring principle, but a set of chemical compounds related to the needs of the plants (glycosides, pectins, proteins, essential oils, vitamins, minerals, carbohydrates) and, concerning elderberry, within each plant source, even belonging to the same family and genus, anthocyanins vary in concentration and chemical structure [30–33]. Usually, when molecular spectroscopies are chosen, the lakes are analyzed by using SERS (Surface Enhanced Raman Spectroscopy), but this involves a direct contact with the original work of art, that is not permitted by the policy of the Italian Ministry for Cultural Heritage. Elderberry, due its usage in medicine and in food, has been thoroughly characterized, in its single components, and spectra from pure or purified compounds — usually dispersed in water solution — are available in literature [34–36], but the direct characterization from a lake applied on a writing support had never been performed before. Fig. 13 reports the Raman spectra of the laboratory elderberry lake compared with a typical spectrum collected from an original mauve area. As can be seen there is a perfect correspondence in the characterizing bands between the two spectra. The enlargement of the bands in the 1200–1600 cm−1 region in the spectrum collected from the original dye is due to the presence of a small amount of carbon black in the original dye, as can be seen in Fig. 13, where the spectrum of carbon black is plotted in the region where the characteristic bands are located. The two intense peaks at 981 and 1009 cm−1 in the elderberry lake are related to the presence of aluminum sulfate, necessary for the manufacturing of the lake.
Fig. 11. Raman spectra of the faded ink the book “Libretto di appunti e memorie del Padre Francesco Zazzera”, before and after removal of the superficial layer of jarosite, which spectrum is reported for comparison. The inset shows on the top the aspect of the faded ink and bottom how it appears after the first jarosite removal attempt. The readability has been recovered. Spectra are arbitrary stacked for a better visualization.
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Fig. 12. Detail of the volume III.4, belonging to the Archive of the Congregazione dell'Oratorio in Rome; a) black inks turned to yellowish-white; b) SEM imaging (BSD in variable pressure) showing the presence of jarosite crystals deposits on the ink surface; crystals presented a typical rhombohedral shape; c) EDS spectra of a crystal; and d) SEM High vacuum imaging on metalized sample: the crystal appeared associated to a biological biofilm, made of both fungal and bacterial structures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A further validation of our attribution was obtained by using FORS (Fiber Optics Reflectance Spectroscopy) at the physics laboratory of Icrcpal. Fig. 14 reports the spectra obtained from the original color and from two elderberry lakes applied on parchment samples by using gum arabic or eggs as binding material. Table 2 reports the position of the peaks. As can be seen, the peaks of the three samples arise at the same wavelength, but the curves obtained with the two binders have a different slope in the 600–800 nm region. In this region the original color presents the same slope of the laboratory sample obtained by mixing the lake with gum arabic. This evidence let us suppose the use of this kind of binder in the Codex. In our knowledge this is the first evidence of the use of an elderberry lake in a very ancient manuscript (6th century). A sort of archeological discovery was also done with respect to the past misfortunes that occurred to the codex, by simply analyzing a few traces (Fig. 15). Diffuse spots affected some of the folios that,
Fig. 13. Raman spectra collected from the original mauve color and the Elderberry (Sambucus nigra) Lake prepared in laboratory. Carbon black spectrum is reported to evidence the enlargement of the peaks in the 1200–1600 cm−1 region, due to the presence of carbon black in the original pigment. Spectra are arbitrary stacked for a better visualization. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
when observed at the stereomicroscope in transmitted light, appeared translucent because of the parchment erosion in those areas. Our hypothesis is that, at a certain point during the last century, the Codex Rossanensis underwent a bacterial attack that caused severe damage to a binding that is now missed, and to the initial pages, resulting in the spotted alteration of the codex. The putative bacterial attack degraded the collagen fibers, leaving the parchment perforated and weak, thus requiring the intervention of restorers. After the intervention, based on gelatin application on the first illuminated folios to strengthen them, the degraded areas showed with a glassy appearance. The bacterial attack to the Codex Rossanensis is only a hypothesis because it could not be confirmed analytically (no samples could be taken for SEM analysis, and the size of the manuscript prevented it to be inserted in the SEM chamber), but it is based on comparison with several case studies well documented by Piñar and other specialists in this research field [37–39]. Evidence that strengthen the hypothesis of a bacterial attack to the codex is observable where the purple stains are scattered close to the text written in silver ink. In these areas the stains “avoid” the writing because of copper impurities present in the silver ink that are toxic to the
Fig. 14. FORS spectra of the original mauve lake and two elderberry lakes dispersed in gum arabic or in egg. The inset shows the logarithmic transformation of reflectance for the original dye and the elderberry lake in gum arabic in characterizing 450–650 nm region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Peaks position (nm) in FORS spectra for the original pink-mauve lake and the elderberry lake dispersed in two different binders (gum arabic and egg). Original mauve color peaks (nm)
Laboratory elderberry lake—gum arabic peaks (nm)
Laboratory elderberry lake—egg peaks (nm)
λ1 λ2 λ3
λ1 λ2 λ3
λ1 λ2 λ3
496.0 ± 1.0 524.1 ± 0.8 564.0 ± 0.2
496.0 ± 1.0 525.1 ± 0.7 564.2 ± 0.3
496.0 ± 0.9 525.1 ± 0.8 564.2 ± 0.2
bacteria. The same phenomenon has been documented in other case studies [37–39]. These bacteria (phylum Actinobacteria) play a major role in the deterioration of ancient parchments. They are filamentous halophilic bacteria known to produce collagenases that are capable of destroying collagen by their hydrolytic activity. They are alkaliphiles and inclined to develop on parchment that, during its manufacture, is processed with alkaline materials such as lime and chalk [38,39].
4. Conclusions The link between the case studies presented in this paper is the discovery, in objects of historical interest, of products or compounds that
Fig. 15. The Codex Rossanensis: a) transmitted light image of p. 15 showing scattered translucent areas where the parchment was thinned by the bacteria; b) detail in natural light of the area of p. 15 showing a direct correlation between the purple stains and the thinned areas (image obtained with a stereomicroscope Leica M16); and c) the typical damage caused by bacteria of the phylum Actinobacteria on a 16th century parchment (showed as comparison respect to the codex). d) Detail in natural light of silver writing on p. 17 showing a peculiar pattern of the purple stains from bacteria that do not overlap onto the ink (image obtained with a stereomicroscope Leica M16). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
are not related with the original materials, but with the vicissitudes of the original documents or their peculiar manufacture. Furthermore, the comparative studies carried out by the laboratories of chemistry and biology highlight the need of a close collaboration between the different competences: many chemical modifications in the original structures can be explained only as a result of biological changes and the latter can be induced by particular chemical compounds present within the documents or in the external environment. To conclude we can say with a smile, that in the analysis of library materials we “have seen things you people wouldn't believe…” [40]. Acknowledgment We would like to thank the colleagues Lorena Botti, Daniele Ruggiero and Maria Teresa Tanasi of the Icrcpal physics laboratory for their collaboration in the FORS characterization of the lakes prepared in the chemistry laboratory and for sharing their FORS results on the original lake. References [1] F. 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