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Patrimonio Histórico

CSIC Thematic Network on Cultural Heritage. Electronic Newsletter COALITION

Newsletter No. 19 January 2010

Index ♦ Microorganisms in museum collections, N. Valentin

2

♦ Analytical protocols for the assessment of biological

damage in historical documents, F. Pinzari et alii 6

♦ The conservation of Papal bulls from the XVth-XVIth

centuries, V. Jurado et alii 13

♦ Contributions to COALITION Newsletter 15

ISSN 1579-8410 Editors: C. Saiz-Jimenez

M.A. Rogerio

Edited by Red Temática del CSIC de Patrimonio Histórico y Cultural Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Apartado de Correos 1052, 41080 Sevilla

Correspondence to:

[email protected]

CSIC Thematic Network on Cultural Heritage and COALITION editors assume no responsibility for statements and opinions advanced by contributors to the newsletter. Views expressed in editorials are those of the author and do not necessarily represent an official position of the Network.

mailto:[email protected]

COALITION No. 19, January 2010

2ISSN 1579-8410 www.rtphc.csic.es/boletin.htm

MICROORGANISMS IN MUSEUM COLLECTIONS N. Valentin Instituto del Patrimonio Cultural de España, Madrid, Spain Introduction The biodeterioration of organic materials is a widespread problem in museums, archives and libraries in regions with warm and humid climates. Moreover, museums, archives and heritage institutions are often located in large buildings and keep large collections consisting of different organic materials. Bacteria and fungi colonize organic materials and are responsible for serious biodeterioration problems in museums and archives, which very often suffer water condensation phenomena and have poor ventilation (Figure 1). The buildings where the collections are deposited also maintain microclimates appropriate for the development of microorganisms in cellulose artefacts (books, textiles, paintings, furniture, wood sculptures), proteinaceous objects (parchment, vellum, leather, mummy skin), and other materials. Microbial development poses two serious risks: • The metabolic production of organic substances that interact with the material. These substances include pigments, enzymes (cellulases and proteases), organic and inorganic acids, chelating agents and other biochemical substances. • The presence of detrimental microorganisms and toxins which pose health risks to conservation personnel and visitors. In the past, the relative humidity of the air was considered as the main indicator of biodeterioration risk and it was usually measured in combination with temperature. More recently, water activity is being used as a measure of how efficiently the water present can take part in a chemical (physical) reaction. Water activity (aw) is defined as aw=p/po, where p and po are the partial pressures of

water above an object and a pure solution under identical conditions respectively. The risk of microbial contamination of a material can be evaluated by the water activity number (aw). It has been reported that most microorganisms can grow in the aw range of 0.6-0.98 (Caneva et al. 1991). Poulsen and Lindelow (1978) indicated that a value of 0.75 allowed fungal growth. According to Florian (1993), bacteria require aw above 0.95, while fungi need a lower aw (commonly 0.70-0.85) which is the usual range where Aspergillus niger, A. glaucus, A. nidulans, Fusarium oxysporum and Penicillium commune grow. However, Pitt (1975) reported xerophylic growth of fungi at aw

0.60. When art materials are exposed to appropriate ventilated environments fungal growth is restricted.

Figure 1. Development of bacteria and fungi on paper. Microorganisms isolated in museums and archives A total of 16 historical buildings located in different climatic regions of Spain were analysed to detect cellulose and protein-degrading microorganisms. Non-destructive and surface samples were taken from objects made of cellulose (paper, cardboard and textiles) and protein materials (parchment, leather, mummies and silk textiles). Air samples were also analysed. Classical methods were used to identify the microorganisms.



Tables 1 and 2 shows the most common taxa and a few representative species of fungi and bacteria isolated from the environment and from objects. The results showed no significant specificity of microbial strains with respect to particular micro-environmental or climatic conditions. The data revealed a high environmental contamination by the fungi Aspergillus, Cladosporium, Penicillium, Geotrichum, and Alternaria which were isolated in poorly ventiIated environments (Figures 2-4). These are dangerous to books and documents due to their cellulolytic activity. The fungal community colonizing paper, parchment and leather was similar to that isolated from air samples. Considerable similarities also existed between both summer and autumn microfungal communities analysed, while winter community differed slightly. Table 1. Fungal species isolated in museums and archives (from Filipello et al. 1989, Gallo 1993, Florian 1994, Valentin et al. 2002)

Genera Species Alternaria A. tenuis*, A. solani Aspergillus A. glaucus*, A. nidulans*, A.

flavus*, A. fumigatus*, A. niger*, A. tamarii*

Botrytis B. cinerea Cephalosporium Cephalosporium sp. Cladosporium C. herbarum*, C. cladosporioides Curvularia C. lunata Chaemotium C. globosum* Epicoccum E. nigrum Fusarium F. roseum, F. oxysporum* Geotrichum Geotrichum sp. Gliocadium G. catenulatum Humicola H. grisea Memnoniella Memnoniella sp. Microsporum Microsporum sp. Myrothecium M. verrucaria Mucor M. racemosus Neurospora Neurospora sp. Ophistoma Ophistoma sp. Paecilomyces P. variotii Penicillium P. frequentans, P. commune, P.

notatum, P. brevicompactum* Pestalotia P. oxyanthi Phoma P. betae, P. pigmentovora Rhizopus R. nigricans* Scopulariopsis S. acremonium Sporotricum S. pulverulentum Stachybotrys S. atra Stemphylium S. botryosum, S. vesicarum Trichoderma T. viride Ulocladium U. consortiale Verticillium V. nigrescens *Potential pathogenic species

Table 2. Bacteria species isolated in museums and archives (from Filipello et al. 1989, Gallo 1993, Florian 1994, Valentin et al. 2002)

Genera Species Aeromonas A. hydrophila Acinetobacter Acinetobacter sp.* Bacillus B. subtilis, B. cereus, B.

circulans, B. mesenthericus Bacteroides Bacteroides sp. Celfalcicula Celfalcicula sp. Cellvibrio Cellvibrio sp. Clostridium Clostridium sp. Corynebacterium Corynebacterium sp. Micrococcus M. luteus, M. roseus Microbacterium Microbacterium sp. Nocardia Nocardia sp. Proteus P. vulgaris Pseudomonas P. aeruginosa* Sarcina Sarcina sp. Serratia S. marcenscens Streptococcus S. viridans* Sporocytophaga S. myxococcoides Streptomyces S. albus*, S. fradiae *Potential pathogenic species

Among the bacteria, Bacillus, Micrococcus and Streptomyces were commonly found. Actinomycetes were always present. Yeasts, such as Candida, were very often isolated. Similar strains were found in the environment and in objects. Bacteria of the genera Micrococcus and Bacillus were mostly identified in the air. Streptomyces and Pseudomonas were often found in paper and leather. Actinomycetes were isolated in 60% of air and 75% of materials samples analyzed. They represented potential pathogenic microorganisms (Salkinoja-Salonen et al. 2003). Paper showed higher fungal contamination than air samples. However, in similar studies, Florian (1994) described the opposite, but this discrepancy could be due to the particular environmental conditions, dust deposition, the rate of ventilation or the specific moisture content of each material. Microbial control Fungi and bacteria growing in organic materials are difficult to eliminate. Many toxic products, including biocides, have been used to prevent biodeterioration in materials and the environment. However, these procedures are usually harmful to people and to the objects they intend to de-infest. Alternative methods based on the use of non-toxic treatments e.g. low temperature or low



oxygen environments, air ventiIation systems for decreasing moisture content etc., have recently been under investigation. Low temperature is not an effective method to eliminate microorganisms. Temperatures in the range of -4 to 0ºC have often been used as an emergency method to avoid microbial development in water-soaked books and documents damaged by floods; but this does not eliminate bacteria and fungi. The disadvantage of this method is the potential risk of damage to delicate materials by chemical and physical changes. Temperatures close to -20ºC can eliminate the development of fungal hyphae and hydrated spores. However, dry fungal spores and many bacteria are resistant to very low temperatures.

Figure 2. Microbiological contamination in a library.

Figure 3. Fungi are the main biodeterioration agents of paper.

Freeze-drying has been used for treating water-soaked paper, textiles and wood. It has also been used to eliminate microorganisms in natural history collections, but it leads to

brittleness and other physical changes in many organic materials (Florian 1990). Many studies have reported that a low oxygen environment is an effective way of decreasing or stopping fungal and bacterial growth (Valentin et al. 1990). It is well documented that most fungi involved in the deterioration of cellulose and protein materials are aerobic (Kowalik 1980, GalIo 1993). In contrast, anaerobic bacteria can develop even at very low oxygen levels, though they need a very high aw (above 0.98%). Nitrogen atmospheres can be used to eliminate insects in furniture, textiles, books, mummies and other art pieces. Experimental treatments have also successfully tested the use of low dynamic nitrogen flows (1 to 5 litres/minute) at 50 ± 5% RH to arrest the growth of detrimental microorganisms. This kind of treatment leads to a drastic decrease in Aspergillus niger, Penicillium sp., Cladosporium herbarum, Trichoderma viride and Bacilus subtilis in paper exposed to 0.005- 0.1% oxygen (Valentin 1990, Valentin et al. 1990).

Singh et al. (1995) reported the effects of air temperature and humidity on fungal growth and recommend the use of alternative technologies, including ventilation, to achieve environmental conditions that decrease the moisture content of historic buildings and thus prevent biodeterioration.

Figure 4. Isolation of bacteria and fungi from paper.

Ventilation has been found a useful, simple and safe way of treating infected objects. Further, it is an effective alternative to air conditioning systems. Valentin et al. (2002) demonstrated that fungi and bacteria stop



growing when exposed to low-level but constant ventilation. The minimum air change per hour (AC/h) required to minimise RH fluctuations and decrease microbial growth in both the environment and collection items depends on the air flow ventilation rate, the temperature, and the volume of the room. The data show that air microflora can remain dormant even at a high RH (80%) if an appropriate AC/h is maintained. The contamination of paper materials may, however be arrested even when they have a high water content (in a range of 10-12%) if stable low temperatures and consistently ventilated conditions are maintained. To decrease or stop microbial growth in heavily infested objects, continued ventilation for 3 months with 4 AC/h is effective in a room of 25-50 m2. Conservation strategies including maintenance, microclimate control through the monitoring of environmental conditions (Camuffo 1988) and the estimation of the number of microorganisms in both air (air quality control) and objects, is recommended for the preservation of heritage items (Florian 1994). Serious risks associated with microbial contamination in people involved in conservation have been summarized by Salkinoja-Salonen et al. (2003). They reviewed health hazardous substances produced by bacteria and fungi, methods for detection and provided recommendations for protection. Recommendations The use of biocides causes serious problems and also affects people’s health. In many countries, strict regulations already forbid the use of common toxic chemicals. For this reason, conservation strategies, maintenance, ventilation, microclimate control through the monitoring of environmental conditions and estimation of the numbers and types of microorganisms in the air and collection items is strongly recommended. Low temperatures, inert gases and ventilation methods can be used to avoid microbial germination and the colonisation of organic materials. These procedures are useful and safe alternatives to biocides. However, they are not completely effective at solving dangerous microbial infestations, especially in large

collections damaged in disasters such as floods or following significant changes in RH. Moreover, the architecture and poor maintenance of many historical buildings in hot and humid climates contribute to the development of species that are difficult to control. References Caneva, G., Nugari, M.P. & Salvadori, O. 1991. Biology in the

Conservation of Work of Art. Rome: ICCROM. Camuffo, D. 1998. Microclimate for Cultural Heritage.

Amsterdam: Elsevier. Filipello, V., Caramiello, R. & Mariuzza, L. 1989. Outdoor airborne

fungi: sampling strategies. Aerobiologia 5: 145-153. Florian, M-L.E. 1990. The effects of freezing and freeze-drying on

materials of natural history specimens. Collect Forum 6: 45-52.

Florian, M-L.E. 1993. Conidial fungi (mould) activity on artifact materials. A new look at prevention, control and eradication. Preprints, International Council of Museums 10th Triennial Meeting. Washington D.C. August 1993 2: 868-874. Paris: ICOM.

Florian, M-L.E. 1994. Heritage Eaters. Insects & Fungi in Heritage Collections. London: James & James.

Gallo, F. 1992. Il Biodeterioramento di Libri e Documenti. Rome: ICCROM.

Gallo, F. 1993. Aerobiological research and problems in libraries. Aerobiologia 9: 117-130.

Kowalik, R. 1980. Microorganisms in library materials. Restaurator 2: 99-114.

Pitt, J.I. 1975. Xerophylic fungi and the spoliage of food of plant origin. In R.B. Duckworth (ed.), Water Relations of Food. London: Academy Press, pp. 273-307.

Poulsen, P.K. & Lindelov, 1978. Acceleration of chemical reactions due to freexing. In L.B. Rocalland & G.F. Stewart (eds.) Water Activity Influences on Food Quality. New York: Academic Press, pp. 650-678.

Salkinoja-Salonen, M.S., Peltola, J., Andersson, M.A., Saiz-Jimenez, C., 2003. Microbial toxins in moisture damaged indoor environment and cultural assets. In C. Saiz-Jimenez (ed.) Molecular Biology and Cultural Heritage, Lisse: Balkema, pp. 93-105.

Singh, A., Ganguli, M. & Singh, A.B. 1995. Fungal spores are an important component of library air. Aerobiologia 11: 231-237.

Valentin, N. 1990. Evaluation of bacterial contamination on art materials by membrane filtration and epifluorescence microscopy. International Biodeterioration 26: 369-379.

Valentin, N., Lidstrom, M. & Preusser, F. 1990. Microbial control by low oxygen and low relative humidity environments. Studies in Conservation 35: 222-230.

Valentin, N., Garcia, R., de Luis, O. & Maekawa, S. 2002. Air ventilation for arresting microbial growth in Archives. Quatriémes Journées Internationales d’Etudes de l’ARSAG: 139-150. Paris.

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ANALYTICAL PROTOCOLS FOR THE ASSESSMENT OF BIOLOGICAL DAMAGE IN HISTORICAL DOCUMENTS

Flavia Pinzari1, Mariasanta Montanari1, Astrid Michaelsen2 and Guadalupe Piñar3

1Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Laboratorio di Biologia, Via Milano 76, 00184 Rome, Italy. E-mail: [email protected] 2Department of Microbial Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 3Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Abstract Biodeterioration of paper and parchment in ancient books and documents represents a cause of great concern for libraries and archives all over the world. The study of the mechanisms underlying the microbiological attack of historical materials has been widely practiced and still represents one of the main focuses of those institutions and laboratories that are involved in cultural heritage conservation. Many studies on the role of micro-organisms in the defacement of cultural heritage utilise standardised laboratory models to establish, under controlled conditions, which species of fungi or bacteria colonise a given substrata, and therefore do not raise the problem of working with objects of art that cannot be cut, sampled, or subjected to invasive analysis. The aim of this paper is the discussion of the procedures required for a conservative approach to the evaluation and description of the damage and organisms responsible for the biodeterioration of valuable books and works of art made from or supported on paper.

Introduction Microorganisms can be responsible for the destruction of cultural heritage, together with several environmental conditions, ageing and the chemical structure of substrate. There is a number of reviews giving a comprehensive picture of the role of microorganisms in the degradation of art objects, such as paintings, stone, wood, paper, masonry, leather, parchment, glass and metal (Bock and Sand 1993, Cappitelli and Sorlini 2005, Ciferri 1999, Griffin et al. 1991, Kowalik 1980, Montegut et al. 1991). However, when a biology laboratory is asked to supply a purely diagnostic explanation for a degradative phenomenon occurring in a precious art work, the challenge of obtaining

an informative sample from the object without damaging it becomes a real operative problem (Montanari et al. 2006). To this should be added the problem of the viability of micro-organisms and their culturability (Amann et al. 1995), which often makes the diagnosis a matter of sheer luck. In fact, as established in environments other than cultural heritage components, the microbial communities found are much more complex and bio-diverse than would appear according to classic culturing methods (Ward et al. 1990). Microbial investigations based on cultivation strategies cannot be regarded as being reliable in terms of reflecting the microbial diversity present in art works samples. When molecular biology techniques have been applied to cultural heritage environments, new spoiling taxa and unsuspected microbial consortia were found to be involved in the discolouration and biodeterioration of paintings and monuments (Piñar et al. 2001 a,b,c, 2002, 2003, Rölleke et al. 1996, 1998, Schabereiter-Gurtner et al. 2001 a,b,c, 2002 a,b, 2003). The routine work of describing and cataloguing which organisms are causing the specific damage on a given object of art suffers, on one hand, from a lack of standardised methodologies and informative techniques, and requires a set of theoretical instruments for operating at maximum levels and without error. On the other hand, it is somewhat dangerous to establish rules and define strict procedures because most of these can result in being too invasive or not informative when applied to a particularly valuable historical object. The degree of invasiveness in sampling an object of historical value depends on several factors like, for example, the size of the object, the presence or absence of detached fragments, the position of the damage with respect to the valuable parts of the object itself, the heterogeneity of the constituting materials, and the further work that restorers might have to carry out on it. In this paper we describe different sampling techniques and instruments that can be employed to obtain information on damage and damaging agents that can affect ancient papers. The aim is a discussion of the procedures required for a conservative approach to the



evaluation and description of the damage and the organisms responsible for biodeterioration in valuable books and works of art made from or supported on paper and parchment (Figures 1 and 2).

Figure 1. An example of ancient paper discolouration due to a fungal attack.

Our rationale is that a knowledge and thorough understanding of the materials from which a volume was made, together with the identification of original damage are fundamental requisites prior to carrying out any restoration work on it. Nevertheless, the identification of the agent responsible for any phenomenon observed can be very complicated and possibly require the use of multiple techniques suited to the specific object on a case by case basis.

Figure 2. Ancient parchment biodeterioration causing a very peculiar purple stain.

Sampling One of the main problems encountered in the biological diagnosis of cultural heritage is the execution of non-invasive sampling. The study of objects of cultural heritage should be carried out, if at all possible, without modifying the objects themselves, especially if these are of small dimensions. In some cases invasive sampling can be performed, but only following specific rules. The procedures required for sampling valuable books and works of art made from paper cannot easily be standardised, but a sort of ethical protocol for managing some valuable objects should be suggested. Some fundamental rules for good practice are as follows: a. Classic culturing, sampling methods and innovative techniques must be specially adapted on a case by case basis to best suit the particular situation that materials found in our cultural heritage present. b. The application of a destructive method should never be considered if there is an alternative, non-destructive method that will accomplish the same research ends. The method to be chosen is that which will call for the smallest possible sample. c. The use of a destructive method is permissible only on fragments that cannot undergo conservation or cannot be reunited (for example, fragments from the margins of pages, bore dust produced by insects, parts that will certainly be eliminated during restoration, such as parts of the binding or cover leaves). d. No fragments should be removed if this action may cause further damage to the object or a loss of information for future research, like in the written areas of the leaves. e. In respect to the sampling of microbial elements, like fungal fruiting bodies and mycelium found on illuminations, the smallest possible samples should be removed, except in the case of important objects which must not be sampled at all. f. For each sample removed, a written note should accompany the object in its “conservation history”, indicating the research



establishment to which the sample was taken and analysed, the name of the laboratory, and the name of the person who executed the sampling and the analysis procedures. g. The sampling procedure should be accompanied by photographs of the object before and after the removal of material and a description of the method used for sampling, also stating the exact part of the object from which the sample was taken. Non invasive sampling Different types of swabs and adhesive tapes can be employed in sampling fungal or bacterial elements from paper (Figure 3). The choice of the most suitable technique should be based on the alteration to be diagnosed. For example, if there are dry and dusty fruiting bodies and free spores emerging from the substrate, a plastic swab might be electrically charged and therefore obtain a not representative sampling of the damaged area. A wooden-cotton swab is most suitable. The use of a cotton swab slightly moistened in a salt solution (i.e. Ringer or Czapek inorganic solution as described in Samson et al. 2001) can be useful for the recovery of spores and elements of microbial colonies from deteriorated paper or parchment fibres, but the moisture can damage the substrate by creating stains. Removable adhesive tape can be used to collect samples of fungal mycelium and sporulating structures from deteriorated paper (Samson et al. 2002). The choice of a tape that requires little effort to remove from surfaces is obligatory due to the fragility of paper and to the fact that a tape with a strong adhesive would strip too many cellulose fibres.

Figure 3. Direct sampling using cotton swabs. The fungal or bacterial structures taken with the swabs can be directly smeared on agar nutritive medium to be checked for viability.

Figure 4. Light microscopic examination of mounted slides carrying adhesive tape samples. Fungal structures that could tentatively be attributed to an Aureobasidium species, growing around a linen fibre (Olympus AX60 microscope).

Figure 5. A sampling procedure with the use of adhesive tape for the recover of fungal structures from paper.

The tape can be directly observed through a microscope after diaphanisation with cedar oil to check for the possible presence of fungal structures (Figure 4). To perform this type of sampling, a 3-4 cm strip of tape is looped back on itself, with the adhesive side facing outwards, and held between the thumb and index finger, or using the thumb and index of both hands as shown in Figure 5. The adhesive side is then pressed gently on the stain or the surface of paper or parchment. The aerial hyphae, conidiophora or fruiting structures, together with a few damaged fibres of the substrata cling to the sticky surface and can be gently pulled from the mat. This method may not be suitable for the study of very fragile materials (i.e. Japanese



paper, or acidic depolymerised paper, felted paper, etc.) or damp materials. Invasive sampling Blades can be used to scratch the paper’s surface where there is evidence of the presence of fungal structures. Needles can be used to assist in the collection of powder obtained through the use of blades. Some fragments of paper (2-3 mm2 width) can, in some cases, be collected, possibly from the margins of the most degraded pages using a watchmaker’s tweezers. Paper samples should be taken only from those margins of the sheets and areas where invasive sampling is not endangering the value of the object itself (Figure 6).

Figure 6. A sampling procedure with the use of needles, performed under a safety cabinet, aimed at the removal of small parts of damaged paper.

Figure 7. A variable pressure SEM analysis (ZEISS EVO 50) of paper fragments sampled from a biodeteriorated document. No metallization was used. Paper fibres and fungal conidia can be observed.

When micro-invasive sampling is necessary, the use of such very small samples must be

optimised to obtain informative results. This is the case with SEM/EDS observation of small samples of damaged ancient paper. In some cases it is possible to make a diagnosis using this technique, especially when classic culturing methods yield no results (Figures 7 and 8). In the specific case of some filamentous fungi, the fruiting body shape, and the conidia size, shape and ornamentation can lead to identification at least at the genera level (Florian 2000, Samson and Pitt 2000). Paper powder or small fragments can be used for direct inoculation on to agar plates containing highly specific media, or used to perform more specific culturing methods.

Figure 8. A High Vacuum SEM (ZEISS EVO 50) micrograph showing bacteria growing on the surface of damaged parchment. The small sample, taken from the margin of a manuscript was sputtered with gold before SEM-observations.

Culturing methods Microbiological research carried out in the field of cultural heritage until today has mainly been based on classical culturing methods. Although these techniques are often helpful for demonstrating the importance of micro-organisms in some deterioration processes, most of them are not suitable for solving all the problems encountered and for an effective impact evaluation. 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 it is now generally accepted that cultivation methods recover less than 1% of the total micro-organisms present in environmental samples (Amman et al. 1996, Ward et al. 1960). Nevertheless, cultivation techniques should be performed paying attention to the specific metabolic and physiological requirements of the species



affecting the substrata that support library materials of cultural heritage 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 fruiting bodies (useful for their identification) (Florian 2002, Samson and Pitt 2000). Moreover, a direct smearing of the cotton swabs on to solid agar medium very often doesn’t cause a colony to develop, because the spores and mycelium fragments picked up by the swab are often nutrient starved and dehydrated. Moistening the dry swab in water or in a nutritive or mineral broth for a few hours prior to agar inoculation can allow the reactivation of dormant spores. When fragments of deteriorated materials are available, a type of culture that is useful for discovering whether the microbial strain is a paper spoiler or just a contaminant can be performed. In this procedure, commonly referred to as “multi-point inoculum”, small pieces of paper are washed in sterile water and divided into 25 or more sub-samples that are inoculated directly on to solid nutritive agar distributed according to a geometric scheme (Figure 9).

Figure 9: Paper powder or small fragments can be used for direct inoculation on to agar plates according to a “multi-point inoculum” procedure. Pieces of paper are washed in sterile water and divided into 25 or more sub-samples that are inoculated directly on to solid nutritive agar distributed according to a geometric scheme.

The frame consists of a grid with 25 nodes spaced out equally. The development of the same fungal or microbial organism in most of the 25 points allows for a statistically significant attribution of the damage to it (Dix and Webster 1995) (Figure 10). When different species develop from the 25 inocula

it is not possible to place the isolated strains into a strict relationship with the damage observed. Following the isolation of a strain suspected of being the responsible for damage, the artificial reproduction of the observed damage on sample paper achieved by inoculating the isolated strain can represent a useful counterproof (Figure 11).

Figure 10. “Multi-point inoculum” procedure. The development of the same fungal or microbial organism in most of the 25 points allows for a statistically significant attribution of the damage to it.

Figure 11. The isolation from an ancient paper of a fungal species (Chaetomium globosum Kunze) and the counterproof of its “pathogenesis to paper” by the re-inoculation of the isolated strain on a paper sample.

Molecular methods In the last years, Molecular Biology is developing at a really fast pace. Many of the molecular techniques available can be readily applied to the study of cultural assets. Molecular techniques allow studying microorganisms from their DNA, RNA and proteins. DNA based techniques allow the identification of single bacterial species in sample material without the cultivation of the organisms (Amann et al. 1995). Most of the experiments carried out in this field so far are based on ribosomal sequences, which are used as phylogenetic markers (Woese 1987).



The ribosomal sequences are present in all organisms and they contain variable and highly conserved regions which allow to distinguish between organisms on all phylogenetic levels. In addition, a lot of data exist in the databases (Maidak et al. 1999), which can be used to compare the DNA-sequences of unknown microorganisms and allow a phylogenetic identification. The extracted DNA can be used as a template to amplify ribosomal gene fragments with primers for universal sequences by PCR (Polymerase Chain Reaction). The PCR amplified fragments can then be cloned. The result of such strategy is a clone library, containing ribosomal sequences as inserts. By sequencing individual inserts and comparing the obtained sequences with sequences present in databases, it is possible to identify the phylogenetic position of the corresponding microorganism without their cultivation.

Figure 12. A gel showing the DGGE (ITS specific amplified sequences) of bacterial communities inhabiting ten samples from different biodeteriorated documents.

There are few works focusing on the investigation of the fungal flora responsible for the biodeterioration of paper materials by applying molecular techniques (Di Bonaventura

et al. 2003, Michaelsen et al. 2005, 2006, 2009, Pangallo 2009, Rakotonirainy 2007). In Michaelsen et al. (2005, 2006, 2009) culture-independent molecular methods were used to identify fungal communities on paper from different time periods and quality. As already applied for environmental fungal communities (Jackson et al. 1999, Jasalavich et al. 2000), nucleic-acid-based strategies targeting rRNA-encoding regions were selected for essays studying the community structure of fungi on biodeteriorated paper. Pure fungal strains were compared to infected material, according to DNA extraction and PCR conditions, as well as the setting of fingerprinting techniques as DGGE (Denaturing Gradient Gel Electrophoreses) analysis (Figure 12). Several extraction methods were tested. An ideal DNA extraction protocol adapted for paper/parchment samples should allow: a) to obtain good results with very small samples; b) to be able, by washing treatments previous to extraction, of distinguishing between the microorganisms responsible of the deterioration and the polluting spores coming from dust deposition (Michaelsen 2006). According to Michaelsen et al. (2006) the bead-beating homogenisation of samples showed best results in fungal DNA extraction from paper materials of different compositions and ages. It rendered a pure DNA able to be directly amplified without the need for any extra purification step. However, the problem still remains that no single method of cell lysis is appropriate for all fungi potentially colonising paper-made art objects. The PCR amplification of the ITS1 region, followed by fingerprinting discrimination of fungal sequences in DGGE analysis is a reproducible and powerful technique for the visualisation not only of viable fungi, but also of formerly active fungi, which could have had an important role in paper biodeterioration processes. Other molecular techniques have been applied to the study of the microflora inhabiting indoor artworks, like in Pangallo et al. (2009) where to type the bacterial and fungal isolates, 2 PCR methods, repetitive extragenic palindromes (REP) and random amplified microsatellite polymorphisms (RAMP) were used.



In Rakotonirainy et al. (2007) the two Internal Transcribed Spacers and the 5.8 S gene (ITS1-5.8S-ITS2) from the nuclear ribosomal DNA were amplified, cloned and sequenced. Following a preliminary treatment with cellulase from Trichoderma reesei, DNA extractions were successfully achieved directly from paper samples. From 22 selected stained spots from a book dating from the 19th century, 8 extracts of genomic DNA were entirely analysed, which yielded 145 sequenced clones. Conclusions Prior to any restorative operation, books, like other objects of cultural heritage, should always undergo an evaluative study (from the chemical and biological point of view) of the damage they present. The sampling represents a very important moment and should never be improvised or performed with inadequate instruments. Although most of the information needed to detect biological damages could be obtained by means of the simple methods as some described in this study, new types of diagnostics could result in more exhaustive tests. Traditionally, microbiology research carried out in this field was mainly based on classical cultivation methods. Although these investigations were helpful to demonstrate the importance of microorganisms in deterioration processes, most of them were not suitable for sustained impact evaluation. The results obtained covered only those few organisms that could be cultivated. It is generally accepted that cultivation methods recover less than 1% of the total microorganisms present in environmental samples (Amann et al. 1995, Ward et al. 1990), and that only about 5% of fungal species can be described owing to culture limitations, misidentifications in culture collections, and unexplored habitats (Hawksworth and Rossman 1997). Besides the very new and fascinating results that can be obtained by molecular approaches, at the purely diagnostic level, there is still a need for unsophisticated and inexpensive biological methodologies that could result in exhaustive tests, being performed on very small samples, and able of defining the cause and degree of damage. Acknowledgements The molecular analysis included in this project and A. Michaelsen were financed by the

Austrian Science Fund (FWF) within the framework of project P17328-B12. G. Piñar was financed by the “Hertha-Firnberg-Nachwuchsstelle (T137)” from FWF. References Amann, R.I., Ludwig, W. and Schleifer, K-H. 1995. Phylogenetic

identification and in situ detection of individual microbial cells without cultivation. Microbiology Reviews 59: 143-169.

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THE CONSERVATION OF PAPAL BULLS FROM THE XVth-XVIth CENTURIES

V. Jurado1, M.P. Pastrana2, E. Porca1 and C. Saiz-Jimenez1 1Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Sevilla, Spain. 2 Departamento de Documento Gráfico. Centro de Conservación y Restauración de Bienes Culturales. Junta de Castilla y León, Spain. The Historical Heritage Foundation of Castile and León, in cooperation with the bishopric of Segovia and the town council of Cuéllar, carried out the restoration of the church of San Esteban and its properties in Cuéllar (Segovia, Spain) (Fundación del Patrimonio Histórico de Castilla y León 2007, 2009). The four sepulchres in the presbytery were restored in 2008. Among them was that of Doña Isabel de Zuazo, the wife of Don Martín López de Córdoba Hinestrosa, an alderman of the corporation of Cuéllar (Figure 1).

Figure 1. Sepulchres of Don Martín López de Córdoba Hinestrosa and Doña Isabel de Zuazo in the presbystery of the church of San Esteban.

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Together with the body was found a series of printed documents from the XVth to XVIth centuries, most of which were bulls of indulgence. Many of the printed documents dated from before 1520, some before 1501, and the oldest (and best-conserved, being on parchment) was from 1484; the most-recent dated from 1535. In total, this invaluable find comprises 45 documents on paper, various fragments, two documents on parchment, and a small prayer book. Their value is immense, because they reveal the inception of printing in Castile and the uses and religious ideas of the time ― the end of the Middle Ages and beginning of the Renaissance.

Figure 2. Bull of indulgence found in the sepulchre of Doña Isabel de Zuazo.

The find is unusual because of the conditions under which the documents have been conserved ― a burial of six centuries ago.

The Centre of Conservation and Restoration of Cultural Properties of the Board of Culture and Tourism of the Junta of Castile and León was in charge of the recovery and conservation of the documents, which presented a high level of microbial colonisation (Figures 2 and 3). A microbiological study of the documents was carried out in the Institute of Natural Resources and Agrobiology, using techniques of molecular microbiology, together with a study by scanning electron microscopy. This report presents some of the results obtained (Tables 1 and 2), indicating the presence of bacteria and fungi on the bulls. Table 1. Phylogenetic identification of bacterial sequences detected in five bulls

1 2 3 4 5 6 7

F260209 ND ND ND 96% ND ND 4%

IZ10.1 ND ND ND ND 100% ND ND

IZ5.1 84% 8% ND ND 8% ND ND

IZ26.1 83% ND ND ND 3% ND 14%

IZ35.2 ND ND 18% ND ND 74% 8%

1. Clostridium sp.; 2. Moraxella sp.; 3. Nocardiopsis sp.; 4. Rickettsiella sp.; 5. Sporosarcina sp.; 6. Saccharopolyspora sp.; 7. Others <5%; ND, not detected

It is remarkable the presence of species of the genus Clostridium, a group of anaerobic bacteria. Clostridium forms part of the gastrointestinal tract of humans and animals (Wang et al. 2005). Vass (2001) reported that human decomposition is associated with anaerobic fermentation and saponification from fats is accelerated by the post-mortem invasion of tissues by bacteria, especially putrefactive species such as Clostridium. The genus Rickettsiella is a pathogen from a wide variety of arthropods, although our sequences were very similar to those of clones obtained from samples of human skin (Penn et al. 2001). The fungal genera Penicillium, Cladosporium, and Epicoccum are found in different habitats, including the soil (Michaelsen et al. 2009), although they are closely related with the degradation of cellulose and paper.

Table 2. Phylogenetic identification of fungal sequences detected in five bulls

Penicillium sp. Epicoccum nigrum Cladosporium cladosporioides F260209 100% ND ND IZ10.1 ND ND ND IZ5.1 ND ND ND IZ26.1 ND 98% 2% IZ35.2 ND ND ND

ND, not detected



Bearing in mind that these documents date from the XVth to XVIth centuries, it has to be recognised that ― under the burial conditions ― their state of conservation is surprising. The microorganisms detected, most of which are associated to the human body and have cellulolytic activity, grew initially under suitable conditions of nutrients and moisture, in the presence of a decomposing body. Such microbial activity caused the degradation of the paper’s cellulose fibres and the formation of stains as reported by other authors (Zotti et al. 2008, Mesquita et al. 2009). Subsequently, the limiting of nutrients, and low temperature and moisture slow the growth of these microorganisms, and thus their metabolic activity, explaining the survival of the documents.

Figure 3. A damaged bull with black, brown and white spots. Fragments of paper were collected using a cotton swab and tweezers.

The re-exposure of the bulls to higher levels of moisture and temperature following their discovery could favour the growth of further microorganisms, threatening its conservation. Acknowledgements Funding from the Consolider project TCP CSD2007-00058 and Consejería de Innovación, Junta de Andalucía, PAI, are acknowledged. Figure 1 was kindly provided by the Historical Heritage Foundation of Castile and León. References Fundación del Patrimonio Histórico de Castilla y León 2007. San

Esteban de Cuellar recupera su espacio. Patrimonio 31: 20-24.

Fundación del Patrimonio Histórico de Castilla y León 2009. Sepulcros al descubierto: hallazgos inesperados en San Esteban de Cuellar. Patrimonio 37: 20-26.

Mesquita, N., Portugal, A., Videira, S., Rodríguez-Echevarría, S., Bandeira, A.M.L., Santos, M.J.A., Freitas, H., 2009. Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra. Internacional Biodeterioration & Biodegradation 63: 626-629.

Michaelsen, A., Piñar, G., Montanari, M., Pinzari, F., 2009. Biodeterioration and restoration of a 16th-century book using a combination of conventional and molecular techniques: A case study. Internacional Biodeterioration & Biodegradation 63: 161-168.

Penn, S.G., Hazel, D.K., Chen, W., Rank, D.R., 2001. Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human placenta. Patent: WO 0157272-A

Vass, A.A., 2001. Beyond the grave — understanding human decomposition. Microbiology Today 28:190–192.

Wang, Y.L., Yang, R.H., Ma, A.J., Wang, J.Q., Dong, Z.Y., 2005. Phylogenetic diversity analyse of rumen bacteria using culture independent method. Wei Sheng Wu Sue Bao 45: 915-919.

Zotti, M., Ferroni, A., Calvini, P., 2008. Microfungal biodeterioration of historic paper: Preliminary FTIR and microbiological analyses. Internacional Biodeterioration & Biodegradation 62: 186-194.

INVITATION TO CONTRIBUTE TO COALITION, A LEADING NEWSLETTER COALITION is an open-access electronic newsletter that provides a forum for scholarly research in Conservation of the Cultural Heritage, and related public policy issues. Scientists, conservators, restorers, who share an interest in cultural heritage studies, fostering an interdisciplinary communication between humanities, science and technology, are welcome. In addition to research papers, the newsletter also publishes short communications, technical notes, description of activities in specialised centres, and book review articles.

The editors welcome contributions, as well as proposals for guest edited special issues. As an official publication of the CSIC Thematic Network on Cultural Heritage, all full members receive COALITION as a member benefit. In addition, COALITION is distributed to circa 1,000 subscribers as pdf file, and later uploaded in the RTPHC web site. The distribution system ensures a wide dissemination of the papers. For further information, to view a sample copy, and for submission details, please visit the Journal’s web page at www.rtphc.csic.es/boletin.htm. All contributions should be submitted by e-mail to [email protected]

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