1. Introduction

The first set of Egyptian mummies arrived in Turin following the scientific/commercial expedition undertaken by the traveler and naturalist, Vitaliano Donati, a professor of botany at the University of Turin, at the behest of King Charles Emmanuel III of Savoy between 1759 and 1762. In Egypt, Donati collected some 1,689 scientific samples and objects,1 including eighteen animal mummies. A second group of mummies, many probably coming from the ancient Egyptian capital city of Thebes (modern-day Luxor in southern Egypt), was collected by Bernardino Drovetti, Consul General of France to Egypt, and was bought, for the Turin collection,2 by the Savoy king Charles Felix in 1824.2 In 1888, when the two-volume catalogue of the Museo Egizio was completed by Ariodante Fabretti (1816-1894), Francesco Rossi (1827-1912) and Rodolfo Vittorio Lanzone (1834-1907), it included a total of twelve complete human mummies, nine heads – two of which were associated with a hand – two hands with rings, four pseudo-mummies, and seventy-eight animal mummies. The human mummies entered the collection inside wooden anthropoid coffins, and the inscriptions that grace these coffins specify the names and professions of the deceased they were meant for, supposedly the same people whose mummified bodies were contained in the coffins.3

Finally, thanks to Ernesto Schiaparelli, director of the Museo Egizio between 1894 and 1928, the number of mummies in the collection was further increased. A buying trip to Egypt, done by Schiaparelli in 1900/1901, led to the purchase of more than 1,500 items, including two naturally mummified adult bodies and the mummy of a small child dating to about 3500 BCE. But it is especially since the foundation of the Italian Archaeological Mission to Egypt in 1903 that a large number of human remains was discovered at various sites along the Nile, during many excavation seasons directed by Egyptologists Ernesto Schiaparelli and, later on, Giulio Farina, who were assisted, since 1914, by anthropologist Giovanni Marro.4 A total of 116 mummified or skeletal human bodies and body parts are recorded today in the Museum database as coming from these fieldwork campaigns, but 20 more mummies, 650 complete skeletons, and 144 skulls, presently held in the Turin Museum of Anthropology and Ethnography,5 most likely also derive from the same excavations.

Given the increasing attention among researchers to the mummified human remains held in the Museo Egizio in Turin, in 2016 the “Mummy Conservation Project” was set up, with the aim of incorporating the data and knowledge acquired so far on these human remains in order to develop a protocol for proper museum conservation and display.

The goals of the “Mummy Conservation Project” illustrated in this paper were the following:

2. Research aim

In view of the Museo Egizio and Eurac Research’s common interest in developing know-how to help preserve all the Museo Egizio’s mummies – both those on display and those in storage – special emphasis was placed on testing water activity (aw), a parameter that affects the rate of biochemical decay in organic tissues. This important parameter is significantly affected by temperature and relative humidity in the conservation environment. In parallel to the assessment of this physical parameter, a first microbial survey was performed.

To identify the presence of possible superficial growing fungi, samples of both textiles and skin were taken using non-invasive contact plates.

To obtain further knowledge of the environmental conditions the mummies are presently experiencing, the researchers also took samples of spores present in the air inside the storage room where the mummies were kept, in a display case in the permanent galleries, and in the room where the research operations were carried out.

The aim of this work is to expand on research on the conservation organic objects of historical and artistic interest by assessing both biological and physical-chemical conservation requirements.

3. Material and methods

3.2 Measuring water activity (aw) of the mummies

As regards this specific phase of the project, a total of 106 organic samples were analysed, both of a vegetal nature (bandage linen) and of human origin (fragments of bone, skin, muscles, connective tissue, hair)(Fig. 1).

To measure the water activity (aw) value, the Rotronic instrument, model HygroPalm HP-23-AW-A, was used. Prior to measuring the samples, the HC2-AW and the relative Rotronic HC2-SH measurement probe were calibrated as needed.

Immediately after sampling the mummified organic tissues, the water activity of the samples was examined. The samples were then placed inside special sample holders, previously sterilised. The Rotronic HW4 software version 3.8.0 was used to validate the measurements obtained. Once the PC was connected to the analyser, it was possible, through this application, to set the necessary operating settings in order to obtain the measurement (Fig. 2).

Simultaneously with the water activity (aw) acquisition phase, the temperature and relative humidity values of the environment were obtained. The time required to acquire each sample is approximately 5 minutes. At the end of each acquisition, a validation certificate was produced in form of a PDF file.

3.3 Sampling and cultivation of fungi

Non-invasive sampling using contact agar plates (DG18 and 2 % MEA, Merck) was performed as previously4 described6 on different materials for the cultivation of superficial growing fungi (Fig. 3). The samples were taken from different materials found in the mummies, including soft tissue and textile material. We generated data from 18 mummies from the museum’s storage room “Ipogeo magazzino visitabile” and 6 mummies stored in display cases. Air-borne fungi were collected on DG18 and 2 % MEA plates using an air sampler (SAS) Super-90 (PBI International, Milan, Italy), with a sampling volume of 100 litres (Fig. 4). Fungi collected from the contact plates and air samples were purified by several transfers onto 2 % MEA and DG18 plates.

3.3.1 DNA extraction, PCR amplification and sequence analysis

Pure cultures were identified based on the sequencing of internal transcribed spacer region (ITSI-5.8S-ITSII), a sequence that can be used as a phylogenetic marker. DNA was extracted from the purified cultures using fresh mycelia, as described by Michaelsen et al.7 For the analysis of fungal ITS sequences, we PCR amplified this genomic region using specific ITS primers,8 using the conditions described by Pinar et al.9 To identify the fungi, we compared the obtained sequence material to fungi ITS sequences available in the online public database NCBI, using the BLAST search program.10

4. Results and discussion

4.1 Observations on the results of water activity (aw) analysis

Water activity analysis is a widely-used technique in food storage control.11 Its application in the field of cultural heritage conservation, on mummified biological samples, is innovative and pioneering.

The importance of relative humidity (RH) to conservation is immense,12 and closely linked to the concept of water activity (aw), the parameter proportional to the escaping tendency of the water molecules present in the tissues constituting the mummy. Water activity (aw) is calculated as the ratio of the partial vapour pressure of water in tissues (Pw) to that of pure water (Pw0) at the sample’s surface temperature.13

After a certain time, any substance placed in a sealed5-6 environment reaches a “thermal and hygrometric” equilibrium with the air surrounding it. Accordingly, the substance’s moisture content reaches an equilibrium with the humidity of the environment (EMC). Under these conditions, the value of the air’s equilibrium relative humidity (ERH) corresponds to the water activity (aw) value of the substance.14

From the chemical point of view, tissues constituting mummies can be considered as concentrated “solutions”, and measurement and limitation of water activity (aw) are essential to the conservation process because they influence the life of microorganisms and enzyme activity. The greater the water activity (aw) value, the faster the decay of organic tissue, as synthetically represented in the chart (Fig. 5).15

Relative humidity values higher than 65 %, combined with temperature values higher than 20 °C, promote the development of moulds and accelerate the metabolism of many harmful insects. It is scientifically demonstrated that, with water activity (aw) lower than 0.3, it is possible to inhibit the majority of the causes of biochemical decay in organic tissue.16

A total of 106 mummified samples were subjected to water activity (aw) tests. The results of the analysis are illustrated in Tables 1A and 1B (Tab. 1A and Tab. 1B).

The results of our water activity (aw) examination shows that most of the analysed mummies are in the range between 0.4 and 0.5 (aw) (Fig. 6). No differences were noted, with respect to this type of analysis, between one mummy and the other, even when kept in different environments. This uniformity of results is not surprising, because the same homogeneity was observed in the climatic parameters of the museum’s exhibition environments and display cases, and of the mummy storage room.

In conclusion, observing the results of the water activity (aw) analysis on the mummified samples, reported in tables 1A and 1B (Tab. 1A and Tab. 1B), and comparing them with recent theoretical and experimental studies relative to the conservation of organic mummified finds,17 it can be stated that:

1. the observed water activity levels are consistent with those expectable in the environmental climatic conditions provided by the museum,
2. the analysed mummies are preserved within the safety parameters provided for proper conservation, as they all remain within the ideal range between 0.25 – 0.65 (aw), as shown in Fig. 5.18

4.2 Microbiological analysis

Through the fungal survey applied in this study, we were able to detect and identify different fungal genera and species on skin and textile samples taken from mummies from both the storage rooms and the display cases (Fig. 7). Interestingly, the number of7-9 different fungi detected on the storage room material (n=17) is substantially higher than the ones from the display cases (n=3). In addition, in contrast to the storage room samples, approximately half of the contact plates taken from the exhibition room samples display no fungal growth at all. This result indicates that the exhibition room material has a lower fungal spore and mycelia load in comparison to the material from the storage room. Further comparison of the detected contact plate fungi with the results obtained from air sampling indicates the presence of air-borne fungi in all the material analysed (Fig. 7, asterisks). For example, fungal species belonging to the genus Penicillium spp. were detected both on the material and in the air in the display cases. However, beside this acquisition and exchange of airborne fungal spores and mycelia, there still appear to be indigenous fungal communities present on different material. The fungal communities detected on the storage room textile or soft-tissue material on display clear sample-specific differences (Fig. 7). These differences become even more apparent when we take a closer look at the functional properties of the “material-specific” fungal species detected. Fungi10 such as Bjerkandera adusta, Aspergillus tubingensis, Cladosporium cladosporioides and Cytospora sacculus were only detected on the textile/plant material.19 For example, while sampling a grass mat using contact plates, we only observed the presence of one fungus, Aspergillus welwitschiae, which is known to be involved in the breakdown of fibrous plant material such as sisal20 (Fig. 7). Taken together, almost all the fungi detected in the textile material are commonly found saprophytes involved in degrading plant remains. In contrast, the soft tissue harboured unique fungal species such as Alternaria infectoria, a saprophytic fungus known to thrive on skin tissue.21 In addition, we detected several Penicillium species (e.g. Penicillium chrysogenum) on the soft tissues, known for their proteolytic potential.22 There is therefore a clear indication of the presence of material-specific fungal communities.

5. Final considerations

In sum, our results indicate a still present biodegradational potential in the form of tissue-specific fungal communities (spores and mycelia), which could become a biodegradative risk as soon as the environmental conditions change, for example, at water activity levels above 0.65 (aw), which promote fungal growth.23 It is therefore of the utmost importance to keep the water activity levels of these unique cultural assets under constant control.

6. Acknowledgements

The authors wish to thank their partners for their valuable collaboration, without which it would not have been possible to complete this study project: the director of the Museo Egizio Christian Greco, Sara Aicardi, Susanne Töpfer, Valentina Turina, Biagio Sparacino (Museo Egizio, Turin), Giovanna Cipollini and Paola Bassetti (Eurac, Institute for Mummy Studies), Jagat Narula, Jim & Linda Sutherland, Chris & Heather Rowan, Samuel Wann, Klaus Fritsch, David Michalik, Sean Reardon, Emily Venable, Magdi Yacoub, Adel Allam, Michael Miyamoto (Horus Group), Toshiba Medical Systems Europe BV Netherlands, Gianni Monfardini (Artería S.r.l. Milan), Tiziana Mozzato, Silvia Vieceli (Starhotels Majestic, Turin).

Bibliography

Altschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman, “Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs”, Nucleic Acids Research 25/17 (1997), pp. 3389–402.

Bensch, K., J.Z. Groenewald, J. Dijksterhuis, M. Starink-Willemse, B. Andersen, B.A. Summerell, H.D. Shin, F.M. Dugan, H.J. Schroers, U. Braun, and P.W. Crous, “Species and Ecological Diversity Within the Cladosporium Cladosporioides Complex (Davidiellaceae, Capnodiales)”, Studies in Mycology 67 (2010), pp. 1–94.

Boano, R., R. Grilletto, A.M. Donadoni Roveri, E. Rabino Massa, and E. Fulcheri, “Le mummie del Museo Egizio di Torino: indagini diagnostiche preliminari sullo stato di conservazione”, in F. Barbagli (ed.), Preparazione, conservazione e restauro dei reperti naturalistici: metodologie ed esperienze. Atti dei Seminari ANMS di Pavia, Museologia Scientifica Memorie 3 (2008), pp. 107–11.

Boano, R., G. Mangiapane, M. Girotti, and E. Rabino Massa , “La collezione antropologica egizia del Museo di Antropologia ed Etnografia dell’Università di Torino”, in R. Boano and E. Rabino Massa (eds.), Mummie Egizie in Piemonte. Storia ed attualità in ambito egittologico ed antropologico, Torino 2012, pp. 21-22.

Boano, R., F. Cesarani, G. Gandini, R. Grilletto, E. Fiore Marocchetti, and M. C. Martina, “Mummie e mummificazione”, in Museo Egizio, Modena 2015, pp. 244–51.

Cheftel, J.-C., H. Cheftel, P. Besançon, and L. Didero (eds.), Biochimica e tecnologia degli alimenti, Bologna 1999², pp. 480–84.

Delorenzi, E., R. Grilletto, Le mummie del Museo Egizio di Torino. N. 13001–13026. Indagine antropo-radiologica (Catalogo generale del Museo Egizio di Torino, serie seconda – collezioni 6), Milano 1989, pp. 17–46.

Duarte, E.A.A., C.L. Damasceno, T.A.S. De Oliveira, L.D.O. Barbosa, F.M. Martins, J.R. De Queiroz Silva, T.E.F. De Lima, R.M. Da Silva, R.B. Kato, D.E. Bortolini, V. Azevedo, A. Gòes-Neto, and A.C.F. Soares, “Putting the Mess in Order: Aspergillus Welwitschiae (and not A. Niger) is the Etiological Agent of Sisal Bole Rot Disease in Brazil”, Frontiers in Microbiology 9 (2018).

Dubois, D., M. Pihet, C.L. Clech, A. Croue, H. Beguin, J.P. Bouchara, and D. Chabasse, “Cutaneous Phaeohyphomycosis due to Alternaria Infectoria”, Mycopathologia 160 (2005), pp. 117–23.

EN 15803, Conservation of Cultural Property – Test Methods – Determination of Water Vapour Permeability, European Committee for Standardization (CEN), 2009.

Fan, X., K.D. Hyde, M. Liu, Y. Liang, and C. Tian, “Cytospora Species Associated with Walnut Canker Disease in China, with Description of a New Species C. Gigalocus”, Fungal Biology 119/5 (2015), pp. 310–19.

Fiore Marochetti, E., “Le mummie delle collezioni Donati e Drovetti al Museo delle Antichità Egizie di Torino”, in R. Boano and E. Rabino Massa (eds.), Mummie Egizie in Piemonte. Storia ed attualità in ambito egittologico ed antropologico, Torino 2012, pp. 5-6.

FprEN 16242, Conservation of Cultural Property – Procedures and Instruments for Measuring Humidity in the Air and Moisture Exchanges Between Air and Cultural Property (under approval), European Committee for Standardization (CEN), 2012.

Labuza, T.P., “Sorption Phenomena in Foods: Theoretical and Practical Aspects”, in: Cho-Kyun Rha (ed.), Theory, Determination and Control of Physical Properties of Food Materials, Food Material Science, I (1975), pp. 197–219.

Marro, G., “Scavi Italiani in Egitto e loro scopo antropologico”, XV Congrès International d’Anthropologie & d’Archéologie Préhistoríque, IV Session de l’Institut International d’Anthropologie, Portugal, 21-30 septembre 1930, Paris 1931, pp. 1–8.

Martín, A., M.A. Asensio, M.E. Bermúdez, M.G. Córdoba, E. Aranda, and J.J. Córdoba, “Proteolytic Activity of Penicillium Chrysogenum and Debaryomyces Hansenii During Controlled Ripening of Pork Loins”, Meat Science 62 (2002), pp. 129–37.

Moiso, B., La storia del Museo Egizio, Torino and Modena 2016, pp. 40–55.

Michaelsen, A., F. Pinzari, K. Ripka, W. Lubitz, and G. Piñar, “Application of Molecular Techniques for Identification of Fungal Communities Colonising Paper Material”, International Biodeterioration & Biodegradation 58/3 (2006), pp. 133–41.

Perrone, G., G. Mulè, A. Susca, P. Battilani, A. Pietri, and A. Logrieco, “Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus Carbonarius, Aspergillus Tubingensis, and Aspergillus Niger Strains Isolated from Grapes in Italy”, Applied Environmental Microbiology 72/1 (2006), pp. 680–85.

Piñar, G., D. Piombino-Mascali, F. Maixner, A. Zink, and K. Sterflinger, “Microbial Survey of the Mummies from the Capuchin Catacombs of Palermo, Italy: Biodeterioration Risk and Contamination of the Indoor Air”, FEMS Microbiology Ecology 86/2 (2013), pp. 341–56.

Piñar, G., L. Krakova, D. Pangallo, D. Piombino-Mascali, F. Maixner, A. Zink, and K. Sterflinger, “Halophilic Bacteria Are Colonizing the Exhibition Areas of the Capuchin Catacombs in Palermo, Italy”, Extremophiles 18/4 (2014), pp. 677–91.

Samadelli, M., G. Roselli, V.C. Fernicola, L. Moroder, and A.R. Zink, “Theoretical Aspects of Physical-Chemical Parameters for the Correct Conservation of Mummies on Display in Museums and Preserved in Storage Rooms”, Journal of Cultural Heritage 14/6 (2013), pp. 480–84.

Scattolin Morecroft, A., “The Vitaliano Donati Collection at the Turin Egyptian Museum”, JEA 92 (2006), pp. 278-82.

Shin, M., Oxygen-free Museum Cases (series II – Research in Conservation), Getty Conservation Institute, 1998.

Wang, Y., R. Vazquez-Duhalt, and M.A. Pickard, “Manganese-Lignin Peroxidase Hybrid from Bjerkandera Adusta Oxidizes Polycyclic Aromatic Hydrocarbons More Actively in the Absence of Manganese”, Canadian Journal of Microbiology 49/11 (2003), pp. 675–82.

White, T., T. Bruns, S. Lee, and J. Taylor, “Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics”, in: M.A. Innis, D.H. Gelfand, J.J. Shinsky, and T.J. White (eds.), PCR Protocols: A Guide to Methods and Applications, San Diego 1990, pp. 315–22.