The contribution of microbiological research in the field of book, paper and parchment conservation

New Approaches to Book and Paper Conservation ‑ Restoration

edited by Patricia Engel, Joseph Schirò, René Larsen,

Elissaveta Moussakova and István Kecskeméti

New Approaches

to Book and Paper Conservation ‑ Restoration

edited by Patricia Engel, Joseph Schirò, René Larsen,

Elissaveta Moussakova and István Kecskeméti

Verlag Berger Horn/Wien

Impressum © 2011 Verlag Berger, Horn/Wien

Alle Rechte vorbehalten – Printed in Austria

Bildrechte den Autoren vorbehalten

Umschlagsbilder: Zsuzsanna Tóth

Umschlag/Layout: Ingolf Schwan

Gesamtherstellung: Druckerei Berger, Wiener Straße 80, A‑3580 Horn

ISBN: 978‑3‑85028‑518‑6

Gefördertvom Bundesministerium für Wissenschaft und Forschung in Wien,vom Bundesministerium für Unterricht, Kunst und Kultur in Wien,durch den Verein Buchstadt Horn – Das Buch im Zentrum,durch das Land Niederösterreich

Bibliographic information published by Die Deutsche Bibliothek

Die Deutsche Bibliothek lists this publication in the Deutsche National biblio grafie;

detailed bibliographic data is available on the Internet at ‹http://dnb.ddb.de›.

British Library and Library of Congress Cataloguing‑in‑Publication Data: A catalogue

record for the book is available from The British Library, Great Britain, and from the

Library of Congress, USA.

7

Content

Ursula Schädler-SaubTheoretical Fundamentals in the Conservation and Restora‑tion of Books: How Helpful are the Theories of Alois Riegl and Cesare Brandi in Practice? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Maria da Conceição Lopes Casanova What do We Need? Education, Ethics, New Values or a Different Perception for the Profession?. . . . . . . . . . . . . . . . . . . . . . . . 45

Weronika LiszewskaAesthetics and Principles of Paper and Book Conservation‑Restoration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Ingeborg UllrichExpiry Date: Unknown – The Experimental Use of Material in Artists’ Book and Installation Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Erich Renhart and Manfred MayerSearching for Traces: Fragments of Former Manuscripts . . . . . . . . . . 111

Nicholas PickwoadLibrary or Museum? The Future of Rare Book Collections and its Consequences for Conservation and Access . . . . . . . . . . . . . . . . . . 113

Spiros Zervos and Dimitra BarmpaInvestigating the Causes of Paper Strength Loss after Aqueous Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Salvador Muñoz ViñasThe Pleural System. An Innovative Approach to Flattening and Lining Large Paper Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Content

8

Petra Vávrová, Petr Kotlík, Michal Ďurovič and Vlasta BrezováDamage to Paper Due to Visible Light Irradiation and Post‑Radiation Effects after Two Years of Storage in Darkness . . . . . . . . . 169

Penelope Banou, Dimitra Barmpa, Maria Giannikou, Giorgos Giannoulis, Ourania Kanakari, Dionisis Roussos and Aggeliki StassinouArchival Records of the New Independent Greek State (mid 19th Century). Where History, Paper Technology and Preservation Meet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Manfred Schreiner and Helmgard HolleUsing X‑ray Radiography in the Documentation of Watermarks on Paper. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Joseph SchiròCopying Presses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Elżbieta Jabłońska Wax Tablets in Polish Collections – Issues Concerning the State of Preservation and Restoration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

René Larsen, Dorte Vestergaard Poulsen Sommer and Kathleen Mühlen AxelssonScientific Approach in Conservation and Restoration of Leather and Parchment Objects in Archives and Libraries. . . . . . . . . 239

Myriam KrutzschIs there a Chance to Safe Egyptian Texts on Leather? . . . . . . . . . . . . . 259

Igor Kozjak and Mirela LeskovacThe Influence of Hydrothermal and UV Treatment on Proper‑ties of Leather Used in Book Conservation. . . . . . . . . . . . . . . . . . . . . . . . . 265

Jedert VodopivecCensus and Analysis of Slovene Medieval Codices. . . . . . . . . . . . . . . 289

Content

9

Theresa Zammit LupiVolume 8 of the Grand Master L’Isle Adam Manuscript Collec‑tion: an Example of Degradation and Pre‑treatment Testing . . . . . . . 317

Zsuzsanna Tóth, Orsolya Koppán, Judit Papp and Marianne ÉrdiRestoration of a Unique Hungarian Medieval Codex Based on Results of Recent International Research and on a New Restoration Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Karin ScheperRefining the Classification of Islamic Manuscript Structures . . . . . . 357

Rodica-Mariana Ion, Sanda Maria Doncea and Mihaela-Lucia IonNanomaterials for Chemical and Biological Restoration of Old Books . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

Rumyana DechevaPreserving the Original Structure of the Medieval Codex During Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

Jolanta Czuczko and Małgorzata Pronobis-GajdzisNineteenth Century Book – Underestimated Beauty . . . . . . . . . . . . . 425

Ekaterina Yu. Andreeva and Svetlana A. DobrusinaSafe Keeping Assessment of Ancient Slavonic Manuscripts . . . . . . . 435

Irina A. Guzner and Andrej Yu. BorodikhinResults of the National Programme – A Complex System of Conservation in Siberia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

Mariana Lucia NesfantuThe Romanian National Library Centre for Pathology and Restoration of Documents – Perspectives and Development Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

Content

10

Gayane EliazyanWritten Heritage Conservation and Restoration in the Matenadaran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

Abdur RasheedRecent Trends in Book and Paper Conservation . . . . . . . . . . . . . . . . . 471

Maja Krtalić, Iva Gobić Vitolović and Damir HasenayPossibilities, Perspectives and Obstacles in Book and Paper Conservation‑Restoration Research: Example of Croatia . . . . . . . . . . 481

Eduard Zaloshnja and Ivan LoliFor a New Policy in the Conservation of Documents . . . . . . . . . . . . . 503

Istvan KecskemétiManaging Archival Collections for Digitisation: Experience from Two Projects of 1.55 and 2.07 Million € . . . . . . . . . . . . . . . . . . . . 507

Simone Pentzien, Andrea Conradi, Robert Koter and Jörg KrügerCleaning of Soiled Paper Model Samples Using Short and Ultrashort Laser Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

Florian Kleber, Markus Diem, Fabian Hollaus, Martin Lettner, Robert Sablatnig, Melanie Gau and Heinz Miklas Technical Approaches to Manuscript Analysis and Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .533

John HavermansGamma Disinfection of Lingo Cellulose Historical Collections. . . . . 559

Flavia Pinzari, Federica Troiano, Guadalupe Piñar, Katja Sterflinger and Matteo MontanariThe Contribution of Microbiological Research in the Field of Book, Paper and Parchment Conservation . . . . . . . . . . . . . . . . . . . . 575

Content

11

Erna Pilch-KarrerNeeds for Paper Research: Now We All Need to Buy SurveNIRs . . . 595

Dirk Andreas LichtblauSurveNIR – a Non‑destructive Evaluation of Material Conditions in Conservation, Actual and Potential Use . . . . . . . . . . . . 603

Marina Bicchieri, Michela Monti, Giovanna Piantanida and Armida SodoApplied Research and Critical Approach: The Proper Way to Deal with “Real” Library Heritage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617

Halina Rosa, Alicja Strzelczyk, Elżbieta Jabłońska, Tomasz Kozielec and Joanna Karbowska BerentStudy of the Adaptation of Bio Cellulose Nano‑Fibers for the Restoration of Historical Paper, Parchment and Textiles . . . . . . . . 629

Samantha SheesleyLascaux Acrylic Adhesives Applied to Paper Conservation . . . . . . . 639

Izabela Zając and Władysław SobuckiUse of WEICON “Sealant & Adhesive Remover” in the Conservation Process of “Lindley’s Plans” . . . . . . . . . . . . . . . . . . 651

Mehmet KonuklarUsing the Triple Mixture of Methylcellulose, Carboxymethyl‑cellulose and Nano‑Micro Calcium Hydroxide Particles for Paper Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665

Yuriy I. Aristov, Ivan S. Glaznev and Larisa G. GordeevaARTIC – A New Family of Humidity Buffers for Libraries and Archives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675

Content

12

Alena Maková, Jarmila Mináriková and Zuzana SzabóováComparison of Fixing Agents for Inks Before Application of Aqueous Neutralization‑Stabilization Treatments on Paper Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693

Benjamin Bartl, Petr Kotlík, Michal Ďurovič and Bronislava BacílkováWorking more Effectively with Bacillus amyloliquefaciens α‑amylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695

Abbas HosseiniThe Story of Reproducing of Shâhnâmeh Baysonghouri . . . . . . . . . . 699

Reni Marcheva-KanovaPreservation of the Library and Archive Collections – Care by Different Specialists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701

Appendix

List of Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

575

Flavia Pinzari, Federica Troiano, Guadalupe Piñar, Katja Sterflinger and Matteo Montanari

The Contribution of Microbiological Research in the Field of Book, Paper and Parchment Conservation

Abstract

Paper is an organic substrate that provides a good medium for the growth of certain fungi, and bacteria, especially cellulolytic strains. The infection and colonisation of paper by fungal spores and propagules take place mainly through air‑dispersion, although direct inoculation of both fungi and bacteria by human handling or by insects and mites, as vectors, can occur. Fungal and microbial development start when there is water available in paper, although the amount of water needed for spore germination and mycelium growth varies according to the fungal and bacterial species involved. In addition, each strain can cause different kinds of degradation phenomena, and particular grades of pa‑per can interact differently with the structures and pigments produced by biological agents. In this contribution, some modern and innova‑tive methods applied to microbiological research for book and paper conservation are presented and discussed. In particular, molecular bi‑ology techniques, enzymatic methods and variable‑pressure scanning electron microscopy and microanalysis are addressed to case studies of historical value. The application of molecular biology techniques on cultural heritage environments has shown that new spoiling taxa and unsuspected microbial consortia are involved in the discolouration and biodeterioration of books and paper‑supported works of art. Moreover, the investigation by means of enzymatic and microscopy techniques of the interaction between the microbial flora, responsible for damage, and the organic and inorganic structural elements in paper proved to be fundamental, in order to understand the mechanisms at play and the degree of alteration found in materials of cultural value.

Flavia Pinzari et al.

576

1 Introduction

Cellulose is the most abundant organic compound found on Earth (30–50 % of plant dry weight) and, in natural environments, represents a major source of energy for microorganisms.1 The storage of books and archival materials inside buildings devoted to their preservation has created unique environments for cellulolytic fungal and microbial species to inhabit.2 Microbial deterioration inevitably occurs on paper of different ages and manufacturing as part of a natural process that human efforts can only delay.

Hueck3 defined the term “biodeterioration” as “any undesirable change in the properties of a material caused by the vital activities of organisms”. Biodeterioration of cultural heritage leads conservators to ask some basic questions which investigators, using not invasive but informative techniques, must answer. The first question is, as ad‑dressed in Hueck,4 following a parallelism between human pathology and biodeterioration, what phenomena can be observed? The answer needs methods capable of documenting symptoms. Microbial degrada‑tion of paper causes different kinds of damage according to the species of organism responsible for the attack. Damage can occur because of mechanical stress or enzymatic action, because moulds can produce a wide range of enzymes (proteinases, gelatinases, cellulases) which are able to destroy the component materials of library and archival col‑lections (Fig. 1).5 Some filamentous fungi frequently associated with paper damage can dissolve cellulose fibres by means of the action of cellulolytic enzymes. Furthermore, fungi produce pigments or organic acids that discolour paper and cause damage to materials of cultural and historical importance made from paper.6

1 Markham/Bazin (1991)

2 Zyska (1997); Florian (2002)

3 Hueck (1968)

4 Hueck (1968)

5 Pinzari et al. (2010a); Sterflinger (2010)

6 Florian (2002)

The Contribution of Microbiological Research …

577

Aging of paper and chemical hydrolysis of cellulose chains can it‑self promote attacks by microbial and fungal saprophytic species. The knowledge and thorough understanding of the materials from which a volume is made, together with the identification of original damage are fundamental requisites prior to carrying out any restoration work on it.

Fig. 1: A printed book affected by purple stains due to a Monascus sp. fungal species.

The second question is what is the cause of the phenomena? The answer can be very difficult because it implies the analysis of the aetiology, with the complication that materials, as a substrate for biodeterioration, are non‑living matter, in contrast to living organisms that react “ac‑tively” to the disease agent. The identification of the agent responsible for any damage observed can be very complex and possibly require the use of technology capable of disclosing useful information. One of the main problems encountered in biological diagnostics of cultural herit‑age is the carrying out of non‑invasive sampling. The study of objects of cultural heritage should be done, if possible, without modifying the ob‑jects themselves, especially if these are of small dimensions. Moreover, in many real situations, the microorganisms that caused the damage are not culturable or no longer viable, even though permanent degra‑dation on the paper has taken place, making the diagnoses problematic when using conventional culture‑dependent techniques.


578

The third question is how do the phenomena develop, and under what external events does the process occur? The description of the deterio‑rative process in all its phases requires a deep knowledge of the biol‑ogy and ecology of the degrading organisms, because all the essential features of damage are defined by the interaction of the noxious agent with the substrate and the environment. To complicate the framework, not all sites where fungal or bacterial growth has occurred show the same kind of damage or stain. This may be a reflection of the range of metabolic attitudes displayed by species as paper spoilage occurs. On the other hand, fungal and bacterial strains with markedly different physiologies can behave in a very similar way towards cellulose fibres and the other paper components, thus resulting in a univocal type of damage. For example, the physiological state of the fungal mycelia, such as the degree of maturity of fungal structures, can lead to differ‑ent effects on a paper’s chemical‑physical properties. Senescent hyphae may release chemicals that young fungal mycelia do not produce, thus causing discolouration and cellulose degradation only under certain age‑related conditions. This situation results in a puzzle which compli‑cates matters for researchers who need to be able to foresee the effects of a degrading agent on a given paper support and, more specifically, in all those fields of application where investigation and diagnostics must be fast and possibly non‑invasive.7

The last but very important question is how can the process be checked and treated? Here the subject becomes thorny. Mass treat‑ments are very controversial, the use of ethylene oxide is to be banned in the EU, gamma rays are known to be harmful to cellulose and other library materials, formaldehyde is harmful to people and protein‑based materials and therefore now forbidden. Topical treatments can hardly be standardised because the interaction with materials and organisms is highly variable and depends on too many factors to be used routine‑ly. Prevention is fundamental, but emergencies and the need for cura‑tive controls are still a reality in many conservative situations. These are the reasons why it is crucial to share renewed efforts for the study

7 Montanari et al. (2006); Pinzari et al. (2009)


579

and evaluation of innovative disinfection procedures with all the scien‑tific community.

2 Microbiological Research

Microbiological research carried out in the field of cultural heritage to answer the questions on the cause of deterioration phenomena is still based mainly on classical culturing methods. Although these tech‑niques are often helpful for demonstrating the importance of micro‑organisms in some noxious processes, most of them are not suitable for solving all the problems encountered and for an effective impact evalu‑ation. Moreover, these studies can provide very little information on the true correlation between certain deterioration processes and micro‑organisms, and often yield poor result on the efficiency of restoration treatments. The results obtainable can cover only those few organisms that can be cultivated, and now it is generally accepted that cultivation methods recover less than 1 % of the total microorganisms present in environmental samples.8 Nevertheless, cultivation techniques should be performed paying attention to the specific metabolic and physiologi‑cal requirements of the species affecting library materials of cultural value. Using the wrong medium can often led to unspecific results or to no results at all. Many of the fungal species that cause damage to paper are, for example, xerophiles, because they require a growth medium with a low water activity (aw) in order to germinate and produce fruit‑ing bodies (useful for their identification).

Some of the most outstanding examples of applications of taxonomy to applied microbiology are in medical, food and public health aspects. Despite decades of investigations in many areas of materials biodete‑rioration, there is still an overall lack of knowledge of the association of specific microorganisms with peculiar types of biodeterioration.

8 Pinzari et al. (2009)


580

Sampling of fungal and bacterial structures from damaged library ma‑terials can be carried out using the following methods:9

• Swab sampling (Fig. 2), where sterile cotton swabs are wiped across spots showing visible damage, transferred to the lab in sterile tubes and then used in culturing techniques to obtain the in vitro growth of microorganisms for further identification.

• Adhesive tape sampling, where suitable transparent adhesive tape is gently pressed over the fungal or bacterial spots, to collect cells, spores, mycelia and fruiting structures. Tapes should be used asep‑tically and transferred in sterile bags to be further processed at the lab. Adhesive tape can be used, in fact, for both microscopic exami‑nations and for direct DNA extraction or biochemical tests.

• Nitrocellulose membrane sampling, where small (2 to 6 cm in di‑ameter) sterile membranes of nitrocellulose are gently pressed aseptically over the bacterial or fungal spots and then immediately transferred on Petri plates containing nutritive agar, or used in fur‑ther diagnostic tests.

Fig. 2: a) Sampling of fungal spores from the print “Généalogie de la Royale Maison de Sa-voye”, (XVII Century) by means of a cotton swab; b) the in vitro cultures of the fungus growing on Malt Agar (left), and on filter paper (right): only when it grows on cellulose fibres it produces pigmented structures.

The direct examination of surfaces with a microscope or the analysis of adhesive tapes used to sample the damaged areas can sometimes answer the question on what the cause of the degradation phenomena is (Fig. 3a). Fragments of adhesive tape with fungal or bacterial samples

9 Montanari et al. (2006); Pinzari et al. (2009)

a b


581

can be transferred on glass slides with a drop of cotton blue stain or with a drop of fluorescein diacetate (FDA) solution (20 μg of FDA in 1 ml of phosphate buffer solution pH 7.3).10 Cotton blue staining is used for observations of fungal structures at a white light microscope, while FDA staining provides a qualitative observation of a microorganism’s active structures (Fig. 3b). FDA stained glasses must be observed by means of an epifluorescent microscope, equipped with a filter for blue excitation wavelength at 495 nm. Active structures (positive staining) are assessed in this method by the presence of a greenish fluorescence emanating from the cytoplasm of spores and hyphae, due to the libera‑tion of fluorescein by enzymatic (hydrolytic) cleavage. Samples stained with FDA should be observed after 20–30 min of incubation in the dark at 20 °C. Lundgren (1981) proved the staining efficiency of the FDA‑method in a comparison with Acridine‑Orange‑staining, fluorescein isothiocyanate (FITC)‑staining, and plate counts to study the number of metabolically active bacteria in soil. Nevertheless, some problems with natural substrates presenting auto‑fluorescence (like cellulose) and fungal species with thick‑walled spores can be encountered when the FDA staining method is used with samples from library materials.

Fig. 3: Observation of a Diploöspora rosea Grove (Hughes, 1968) found on a illuminated parchment and sampled by means of adhesive tape; a) the fungus observed in light field without staining at the optical microscope (Leica DM 5000); b) the same fungus observed with optical microscope equipped for epifluorescence after FDA staining. The presence of a greenish fluorescence indicates the viability of the fungus. The bars in the pictures indicate 10 µm.

10 Colgan/Claridge (2002)

a b


582

Correct identification and adequate characterization of microorganisms can in certain circumstances assist in tracing their origin and therefore help in controlling the problem. A better answer to the question of the identification of the causative agent of the biodeterioration phenomena can come from molecular diagnostic methods. The analysis of the ae‑tiology and the identification of the agent responsible for any damage observed on materials can be afforded by extracting and directly ana‑lysing the genetic information of the noxious organisms.

In recent years, techniques based on DNA analysis are providing re‑searchers with additional tools allowing the identification and genetic characterization of microbial taxa. Molecular methods have changed our ways of understanding of the composition, phylogeny, physiology, and function of microbial communities in the environment. Molecular techniques enable the investigation and monitoring of microorganisms from their DNA, RNA and proteins. DNA based techniques allow the identification of single bacterial or fungal species in sample material without the cultivation of the organisms.11 Most of the experiments car‑ried out in this field are based on ribosomal sequences, or ITS regions, which are nested in the rDNA repeat and are used as phylogenetic markers.12 They contain variable and highly conserved regions, which allow the discrimination between organisms on all phylogenetic lev‑els. In addition, data existing in the databases are growing everyday,13 and can be used to compare the DNA‑sequences of unknown microor‑ganisms to obtain a phylogenetic identification. Besides the progresses achieved in the application of molecular techniques on environmental samples, there is still little work focusing on the molecular investiga‑tion of fungal flora, responsible for the biodeterioration of paper ma‑terials.14 In Michaelsen et al.15 culture‑independent molecular methods were used to identify fungal communities on paper from different ages

11 Amann et al. (1995); Piñar et al. (2001a, 2001b, 2001c, 2002, 2003)

12 Ward et al. (1990); Sterflinger/Prillinger (2001)

13 Maidak et al. (1999)

14 Di Bonaventura et al. (2003); Michaelsen et al. (2005, 2006, 2009, 2010); Pangallo et al. (2009); Rakotonirainy et al. (2007); Mesquita et al. (2009); Cappitelli et al. (2010); Principi et al. (2011)

15 Michaelsen et al. (2005, 2006, 2009, 2010)


583

and of different quality. As already applied for environmental fungal communities,16 nucleic‑acid‑based strategies targeting rRNA‑encoding regions were selected for essays studying the community structure of fungi on biodeteriorated paper. The PCR amplification of the ITS1 re‑gion, followed by fingerprinting discrimination of fungal sequences in DGGE analysis is a reproducible and powerful technique for the visual‑isation not only of viable fungi, but also of formerly active fungi, which could have had an important role in paper biodeterioration processes. To be reliable, DNA extraction protocols adapted for paper/parchment samples must work with very small samples and be able, by washing treatments performed before extraction, to help in the taxonomy of the microorganisms responsible for the deterioration, and to distinguish the polluting spores coming from dust deposition.17

3 6 10100 bp

ITS650 pb

Fig. 4: A gel showing the PCR products of ITS specific amplified se-quences from three fungal strains (3 = Alternaria alternata [Fr.] Keissl., 6 = Cladosporium sphaerospermum Penz., 10 = Stachybotrys chartarum [Ehrenb.] S. Hughes) inhabit-ing a biodeteriorated document. On the left, the molecular weight reference is showed.

16 Jackson et al. (1999); Jasalavich et al. (2000)

17 Michaelsen et al. (2006)


584

Once established that the causative agent of damage is biological, the measure of the viability of the noxious microorganism can represent a challenge, especially when only a scarcely representative sampling is allowed on the cultural object. Very sensitive and strong methods are needed that can be used also in the tests that are performed to verify the effects of treatments and curative actions on materials. Among the methods routinely utilised in biodegradation surveys, the measure‑ment of adenosine tri‑phosphate (ATP) concentration is one of the most reliable.

ATP is responsible for coupling intracellular energy‑producing and energy‑requiring metabolic reactions. At the death of the cells ATP is no longer produced and the remaining ATP is quickly degraded by AT‑Pases (enzymes converting ATP in adenosine diphosphate ADP, and adenosine monophosphate AMP). Therefore, the presence of ATP can be used to characterize living organisms. Rakotonirainy et al.18 suc‑cessfully applied the luciferin‑luciferase method for the quantitation of intracellular ATP (adenosine tri‑phosphate) concentration to infected library materials, in order to check the actual vitality of fungi on paper. ATP is contained inside the cells, and in order to quantify its concentra‑tion it is necessary to disrupt bacterial cells and fungal/bacterial walls. The extraction of ATP can be conducted by using dimethylsulfoxide (DMSO) 90 % in TAE buffer (buffer solution containing a mixture of tris‑hydroxymethyl‑aminomethane base, acetic acid and ethylene di‑amine tetra‑acetic acid), preheating DMSO at 100 °C for 1 minute, as described by Rakotonirainy et al.19 The quantification of ATP contained in the samples can be performed by using a detection kit like the EN‑LITEN ATP Assay System (Cod. FF2000, Promega, USA, Madison) and a Luminometer. The accuracy of measurements is usually within 5 %. As an example of application of the ATP test in microbiological diagnostics for paper biodeterioration, the case of the large (3250 × 1910 mm) print “Généalogie de la Royale Maison de Savoye”, 17th century is reported.20

18 Rakotonirainy et al. (2003)

19 Rakotonirainy et al. (2003)

20 Pinzari et al. (2010b)


585

The print was affected by dark stains passing from the supporting canvas to the paper surface. Adhesive tape samplings showed the pres‑ence of fungal structures characterised by a darkly pigmented myce‑lium. The fungus was unable to grow on conventional nutritive agar medium and the ATP test was applied to quickly verify the viability of fungal structures in order to rapidly make a decision on the need for disinfecting treatments.

Figure 5a shows a detail of the print, and Figure 5b reports the his‑togram summarizing the results of the ATP test. Control samples were obtained from areas of the print without stains, while the extracting mixture used on a clean swab represented blank samples. The results showed that fungal structures were alive and active on both canvas and paper.

The analysis of the relationship between the spoiling microorganism and the substrates can be helpful in documenting the symptoms of the degradative attack on the different components of the cultural material. The Scanning Electron Microscopy (SEM) is an invaluable tool that al‑lows the direct observation of samples and can help in gaining knowl‑edge on the association of specific microorganisms with peculiar types of biodeterioration. The SEM technique is highly informative also in the evaluation of the effects of curative treatments directly on materi‑als and organisms. The direct observation of samples obtained from stained areas of materials of cultural value can add to the knowledge on the association of specific microorganisms with peculiar types of biodeterioration.

The development of scanning electron microscopes (SEMs) that can tolerate specimen chamber pressures in the range 1– 20 Torr (0.13–2.7 kPa) while maintaining the electron gun and column at high vacuum (< 10–6 Torr, 0.13 mPa) represented a revolution in many fields of research applied to cultural heritage. The presence of a gas in the specimen chamber allows for imaging of uncoated specimens at high beam energies and, in instruments that can tolerate chamber pressures in excess of approximately 4 Torr (0.5 kPa), wet and liquid samples.21 Variable pressure SEM technique can be successfully applied to the

21 Goldstein et al. (2003)


586

study of ancient manuscripts, inks, illuminated parchments and writ‑ing supports. Several applications in the field of book and manuscript archaeology and conservation are possible: the study of manufacturing materials and processes, the analysis of degradation phenomena, the evaluation of restoration practices or the description of microscopic ef‑fects of chemical treatments.22 VP‑SEM permits the description of the samples’ surfaces without prior preparation by means of metallization, and therefore represents a micro‑invasive methodology or, when re‑ferred to the sample, a non‑destructive technique. Samples can, in fact, be mounted reversibly on a metal stub using double‑sided carbon ad‑hesive tape, or can be arranged on non‑invasive supports that can be as large as the sample‑chamber of the instrument, thus allowing for the observation of whole objects without damage, or with very limited damage.

ATP concentrationRelative Units of Luminescence (RLU)

Canavas Paper Control Blank

50000

40000

30000

20000

10000

Rela

tive

Uni

ts o

f Lum

ines

cenc

e

SwabAdhesive tape

Fig. 5: a) A stained area of the print “Généalogie de la Royale Maison de Savoye” XVII Century; b) histogram summarizing the results of the ATP test performed on the black stains.

As an example of the application of SEM technique in microbiological diagnostics, the SEM micrographs of adhesive tape samples, used to collect fungal structures directly from cultural materials, are shown in Figure 6. Figure 6a represents the Diploöspora rosea Grove found on an illuminated parchment after sampling by means of adhesive tape. This fungus was actively growing on cultural material, as shown in Figure 3. It is a fungus characterised by very slow growth on agar medium, and until now, it has not produced fruiting structures but only sterile my‑celium in culture. The SEM picture shows that there were some bacte‑

22 Pinzari et al. (2006)

a b


587

ria belonging to the Actinomycetales Order (presumably to the Genus Streptomyces) occurring simultaneously with D.rosea. Figure 6b shows the fungi responsible for the black stains defacing the print “Généalo‑gie de la Royale Maison de Savoye” (17th century) shown in Figure 5.

Fig. 6: a) SEM micrograph of a fragment of adhesive tape used to sample a document affected by Diploöspora rosea and bacteria (white arrow); b) SEM micrograph of cellulose fi-bres infected by a cellulolytic fungus with typical dictyospores sampled from the print “Généalogie de la Royale Maison de Savoye” by means of adhesive tape. The bars in the pictures indicate 10 µm.

3 Perspectives

In terrestrial ecosystems, decomposers mostly live in soil and their di‑versity is far higher than that of primary producers. The total world‑wide diversity of soil fungi, the most important microbial decomposers in many ecosystems, is estimated at 0.5–1.5 million species.23 A library or an archive, from the microbial point of view, represents a wide source of organic material that can be colonized and exploited: the diversity of decomposers (which in this case are called biodeteriogens) in human made environments depends on few and scarcely predictable factors. The development and maintenance of a fungal community on a shelf of a library or in a single book depends on the spores that reach the mate‑rial’s surface, the microenvironment (temperature, relative humidity, light), the water activity of the substrate and the casual events that help colonization of materials (insect dispersion, human contamination, ex‑

23 Hawksworth/Rossman (1997)

a b


588

ternal sources of fungal diversity). Besides the hygienic aspects of the argument, some interesting ecological considerations can be raised. A library or a single book can be compared to virgin land that can be reached by some colonizing organisms that behave like pioneering spe‑cies on a bare soil. For Wardle,24 species identity and the composition of decomposers have far greater impact on ecosystem processes than species richness per se. When considering a book or books stored in a closed environment, the colonization and the biodegradation of con‑stituent organic material depends on the species identity and composi‑tion since only a few organisms can exploit the bulk of the substrate. As in natural environments, the diversity‑functioning relationship is driven by the presence or absence of key species,25 niche differentiation and species interaction. A new way to look at paper and book microbio‑logical diagnostics is now possible, thanks to the innovative techniques available, like molecular diagnostics and SEM operating at environ‑mental conditions.

A better understanding of the natural mechanisms that support the establishment of different species assemblages on cultural assets could be of help in defining new control strategies and treatment procedures, thus assuring human and environment friendly approaches to biodete‑rioration problems.

Acknowledgments

The authors are grateful to Prof. Oriana Maggi and Dr. Dario Lunghini for their invaluable support in the characterisation of the Diplo öspora rosea Grove quoted as an example of a spoiling fungus in the arti‑cle. Guadalupe Piñar was financed by the Austrian Science Fund (FWF) within the framework of the “Elise Richter‑Nachwuchsstelle” (V 194‑B20).

24 Wardle (2002)

25 Wardle (2002)


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The contribution of microbiological research in the field of book, paper and parchment conservation

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