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Fungal and bacterial strains were isolated from some Egyptian mummies (from the Ismailia Museum, Ismailiaa, Egypt; the El-Dokki Agriculture Museum in Cairo, Egypt and human mummies in storage at the Ehnasia Museum in Beni-Sweif, Egypt). The biological samples were taken from indoor air of the mummy chamber as well as from the linen bandages and some deteriorated mummies from museum storage. Seven species of fungi were isolated and identified as Alternaria tenuis (9.5%), Aspergillus humicola (23.8%), Aspergillus niger (23.8%), Chaetomella horrida (9.5%), Chaetomium globosum (14.28%), Hormodendrum viride (14.28%) and Penicillium corylophilum (4.9%). It is obvious from the morphological properties and biochemical activity of the microorganisms that they were able to decompose proteins, cellulose, fats and starch, the fundamental contents of a mummy's body. The most prominent bacteria isolated were Halococcus morrhuae (30.76%), Streptococcus pyogenes (23.07%), Micrococcus Kristinae (15.38%), Micrococcus cinereus (15.38%) and Halobacterium pharaonis (13.38). Results show that A. niger, A. humicola, H. viride, and P. corylophilum were very sensitive to the mummification material of natron salt. The growth of P. corylophilum was completely inhibited at all concentrations of benlate and thymol while A. niger and H. viride were completely inhibited at all thymol concentrations and at 100 ppm of benlate fungicide. The growth of H. viride was completely inhibited at 5% of cedar oil. The fungal isolates of A. niger, H. viride, and P. corylophilum were found to produce various amounts of extracellular enzymes (Avicelase, CMCase and cellobiase).These enzymes play an important role in deteriorating linen bandages as well as the mummy’s body. Research Aims The objectives of the present investigation are to study the fungal colonization and exoenzymatic activities of some deteriorated Egyptian mummies, and the effect of mummification materials on the fungal growth to determine the efficiency of these materials in the mummification process. Furthermore, another important aim is to study the effect of some fungicides in preservation of mummies from fungal attack. Introduction Biodeterioration is considered a great factor in the decomposition of the Egyptian mummies, because fungi and bacteria grow and feed with many of their constituent products, such as protein, fats, starch and cellulose, which represent a stable substrate to microbial growth. There are a number of abiotic and biotic factors such as pollution, light, humidity, temperature, microorganisms, insects, etc., that have deteriorating effects on museum materials [1]. Among these, biological agents such as actinomycetes, fungi, bacteria etc., may cause massive damage to museum objects [2]. Studies on indoor aeromycoflora have attracted the attention of several aerobiologists [2-5]. Martinez et al. [6] isolated a total of 469 fungal colonies from 12 mummies that presented deterioration attributed to colonizing fungi. Among the isolated fungi Penicillium, Cladosporium and Aspergillus were found. Most of these fungi have the ability to produce various enzymes (cellulases, amylases, proteases, keratinases, etc.). Cellulase enzymes degrade cellulose found in linen bandages to double sugars and endoglaconases enzymes cut the cellulose chain in a random fashion whereas, exogluconases enzymes successively remove single cellobiose or glucose units from the non-reducing end of the cellulose chain [7, 8]. Many fungi are able to cause zoonotic superficial infections as a consequence of invading keratinize tissues of skin, hair, and nails [9, 10]. Materials and methods Samples Biological samples were taken from a linen-wrapped mummy, from the air of mummy chambers and from deteriorated Egyptian mummies preserved in museum storage as follows: human mummy no. 2520 and 2519 from the Ismailia Museum (Ismailiaa, Egypt); animal mummies No. 35 and 93 from the El-Dokki Agriculture Museum (Cairo, Egypt) and human mummies in storage at Ehnasia Museum (Beni-Sweif, Egypt, see figures 1-2). Sampling of air spora surrounding the above mummies was done using the plate exposure method [11]. Isolation and identification of fungal strains Swaps from linen bandages wrapped mummies were transferred aseptically to potato dextrose agar plates [12] which were used for the cultivation of bacteria and moulds. The Petri dishes were incubated at 28 ± 2 °C for 7 days for fungi and 3 days for bacterial counts. Fungal isolates were later microscopically analyzed and identified [13-15]. The scheme of Buchanan and Gibon [16] was employed in the identification of bacterial isolates. The frequency occurrence of each genus was expressed as the percentage of samples containing a given organism. Effect of some materials of mummification on fungal growth Each of the following mummification materials - natron, myrrh, juniperus, Cinnamomium camphora, Arabic gum, cassia, mastic resin, and beeswax - was separately examined for its effect on the visual growth of some selected fungi isolated from the mummies using PDA plates. 1 ml of spore suspension (approximately 106/ ml) of 7 days old culture was placed in a Petri dish and poured by PDA medium. After solidification 0.2 g of the mummification material was put in the centre of the dish plate (1 cm diameter) and was fumigated by UV light. Dishes were incubated at 28 ± 2°C for 7 days. The developed colonies of various fungal growths were visually determined using the following scale: + (10% growth), ++ (50% growth), +++ (75% growth) and ++++ (100 % growth). Effect of some fungicides on the linear growth of fungi Three kinds of fungicides recommended for use in archaeological field were tested in this experiment at different concentrations in order to ascertain their effect on the growth of fungi and their inhabitation effect. Table 1 shows the fungicides, their chemical name and composition, and the concentrations used based on the active ingredient. The fungicide was added to PDA medium after sterilization, while still warm. Incubation was carried out at 28 ± 2°C for 7 days.
Enzymatic activity of fungal isolates
For studying the enzymatic activity (such as Avicelase and endoglucanase) of the isolated fungal strains, three isolates of the tested fungi were screened for their abilities to produce extracellular enzymes in liquid media with the use of some buffers (0.05 M Citrate-phosphate buffer, PH = 4.8 and 0.02 M Acetate buffer, PH = 5) which contains 0.02 % sodium azide. Two kinds of reagents have been used (Arsenomolybdate reagent (A), Somogyi copper reagent (B). Growth medium and substances Mandels and Weber's medium [17] was prepared, containing (g/L): 1.4 (NH4)2 SO4; 2.0 KH2PO4; 0.3 urea; 3.0 CaCl2; 0.3 Mg SO4.7H2O; 0.005 FeSO4; 0.0014 ZnSO4; 0.0016 MnSO4; 0.002 COCl2; protease peptone (1%); tween 80 (0.1%) and Avicel (1%) with final pH of 5.0. The medium was supplemented individually by cellulose, protein, fats and starch as carbon sources instead of Avicel. Growth in liquid culture Erlenmeyer flasks containing 50 ml of Mandels and Weber's medium with adequate carbon source were inoculated by 5% (v/v) inoculums of the tested fungi (Aspergillus niger (4), Hormodendrum viride and Penicillium corylophilum). The flasks were then incubated at 28± 2°C for 5, 7 and 20 days. After each incubation period the content of each flask was filtered. Culture filtrates were subjected to enzyme assay. Enzyme preparation Extracellular enzymes were prepared by filtrating the culture through filter paper (Wattman no. 1) while intracellular enzymes were obtained by grinding the washed, cold mycelium with sand in a minimum volume of citrate-phosphate buffer (0.05 M, PH 4.8 ), then centrifuging the mixture and using the supernatant as the enzymes’ solution. Enzyme assays Avicelase (1,4-β-D-glucan cellobiohyrolase) was measured according to Seddler and Khan [18] using Avicel cellulose as substrate, while endoglucanase (1,4-β-D-glucanhydrolase) was assayed as carboxymethyl cellulose (CMCase ) according to the method of Mandels and Weber, the resulting reducing sugar, in both cases was measured by Somogyi reagent [19] using glucose as standard. β-Glucosidase was performed by a modification of Bergham and Petterson method [20] where 0.5 ml of enzyme solution was incubated with 0.5 ml of 0.4 % cellobiose in 0.5 ml citrate-phosphate buffer at ph 4.8 for 30 min at 50 °C. The reaction was stopped by heating the mixture in a boiling water bath for 5 min. The enzyme activity was determined by measuring the concentration of the released glucose using glucose – oxidase kit (Bioanalytical Laboratories- Palm City, U.S.A), and an enzyme unit is considered the necessary amount of enzyme to liberate one µmol of the reducing sugar under assays conditions specified above. Results and discussion Survey of fungal and bacterial isolates Seven species were isolated and identified using optical microscopy as: Alternaria tenuis, Aspergillus niger, Aspergillus humicola, Chaetomella horrida, Chaetomium globosum, Hormodendrum viride and Penicillium corylophilum. The frequent occurrence was found to be ranging from 4.7% for Penicillium corylophilum to 23.8% for Aspergillus niger and Aspergillus humicola. The other species could be arranged based on the frequency of their occurrence as follows: Chaetomium globosum and Hormodendrum viride (14.28%) and Alternaria tenuis and Chaetomella horrida (9.5%). There are many reports dealing with the microbial levels, such as Abdel-Kareem et al. [21], Cook and Rayner [22], Darwish and Sahab [23]. Five bacterial species belonging to four genera were identified and classified as: Halococcus morrhuae (30.76%), Streptococcus pyogenes (23.07%), Micrococcus Kristinae, Micrococcus cinereus and Halobacterium pharaonis (15.38%). Effect of some mummification materials on visual fungal growth
The data in Table 2 shows that the mummification materials have different ability to inhibit the mycelial growth of the tested fungal isolates. The five strains of A. niger, H. viride, P. corylophilum and A. humicola were very sensitive to natron salt and relatively sensitive to a Cinnnamomum camphora extract (figure 3). The natron salts had a large effect on the growth of the fungal isolates because it inhibited the fungal growth on a large area, as the NaCl present in natron salt is an historic preservation material. On the other hand, the same fungal isolates were less affected by myrrh and beeswax extracts and the mummification materials of Juniperus, Arabic gum and cassia extracts were found to have no effect on the tested fungal growth. From left to right: Enzymatic activity
Enzyme activity of cellulase production in fungal strains was measured with different substrates (cellulose, protein, fats, and starch) to know the ability of these fungi to produce these enzymes that react with the mummy’s skin and wrappings causing its decomposition. The results shown in Tables 3-5 reveal that all the tested fungi produced various amounts of extracellular enzymes (Avicelase, CMCase and cellobiase). The value of enzyme production depended on the fungal species and age of the culture. The ability of these fungi to decompose casein, cellulose, oil and starch has also been confirmed by other researchers. a) Avicelase enzyme The tested fungi showed feeble Avicelase activity ranging from 0.041 U/ml to 0.115 U/ml for A. niger, from 0.001 to 0.143 U/ml for H. viride and from 0.009 to 0.399 U/ml for P. corylophilum after 5 and 20 days respectively (Table 3). The highest activity of Avicelase was recorded for cellulose following casein substrates.
b) CMCase enzyme Table 4 showed that all the tested fungi produced different values of extracellular CMCase ranging from 0.103 to 0.643 U/ml for A. niger, from 0.013 to 2.050 U/ml for H. viride and from 0.010 to 1.136 U/ml for P. corylophilum. The highest activity of MCase was recorded for cellulose and casein substrates. Many authors [24-27] reported that all tested fungi had the ability to decompose cellulosic materials and produce cellulase enzymes.
c) Cellobiase enzyme
Table 5 shows that the highest cellobiase activity (7.861 U/ml) was obtained from the culture filtrate of A. niger after 5 days on starch substrate and the lower level (4.690 U/ml) on casein. In H. viride culture filtrate the highest value (3.703 U/ml) was shown on cellulose substrate and the lowest level (0.105 U/ml) on oil substrate after 5 days incubation period. High level of cellobiase activity (5.740 U/ml) was obtained in culture filtrate of P. corylophilum after 20 days on casein substrate and lower cellobiase (0.020 U/ml) was showed on oil after 20 days. The production of cellobiase from different fungi was reported by several authors [25, 28, 29, 30].
Effect of some fungicides on the linear growth of some fungi
Table 6 shows the effect of some fungicides as agar amendment on the mycelial linear growth (A. niger, H. viride and P. corylophilum). Data indicate that the antifungal activity of benlate, thymol and cedar oil against fungal growth increased with the growth in fungicide concentration. The linear growth of P. corylophilum was completely inhibited at all concentrations of benlate and thymol while the growth of A. niger and H. viride was also completely inhibited at all concentrations of thymol and benlate at 100 ppm. The growth of H. viride was completely inhibited at 5% of cedar oil. These results are in total agreement with those reported by other researches on several fungi [31].
Cleaning procedures
Removal of microbial stains and growths over the mummies has been carried out using scalpels and soft brushes accompanied with an extraction system, to reduce the spore's contamination in the working area (figure 4). Cleaning started from the middle to the edges to easily collect the fungal growths and dirt particles without the spread of the microbial contamination (figure 5). Isopropyl alcohol has been used to sterilize the cleaning equipment and the surrounding environments during and after cleaning (figures 6-7). White free acid papers have been place below the mummy to better collect the fungal conidia. Benlate fungicide (200 ppm) has been applied on the mummy's wrapping using a sprayer. After cleaning, mummies were covered by polyethylene to protect them from further contamination. For health and safety issues, the conservator must wear a mask, coat, goggles and gloves during cleaning to avoid inhalation of the microbial contaminants (figure 8). From left to right:
Figure 5. Mummy's wrappings after cleaning. Figure 6. Sterilization of the surrounding air during cleaning. Figure 7. Collecting and removal of dirt particles and fungal growths from a mummy. Figure 8. The necessary tools for conservator' health and safety (masks, gloves, goggles). Conclusion After isolation of the microbial species from Egyptian mummies, experimental studies confirmed the efficiency of some of mummification materials on the inhibition of fungal growth, indicating that mummification materials may also function as biocides. The study indicates the ability of the isolated fungal strains from the Egyptian mummies to produce various amounts of extracellular enzymes (Avicelase, CMCase and cellobiase) which may lead to the decomposition of the mummy materials. Benlate, thymol and cedar oil show an ability to inhibit fungal growth. The authors would like to recommend creating better preventive conservation procedures in order to preserve the mummies from future damage. 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Sahaba, Physiological studies on microorganisms isolated from deteriorated from old manscripts, Master Thesis, Faculty of Agriculture, Ain Shams University (Egypt), 1988 [31] A. H. A. Ellil, and E. F. Sharaf, “Growth, Morphological Alterations and Adaptation of Some Plant Pathogenic Fungi to Benlate and Zineb. A New Look”, Journal of Biological Sciences 3(3), 2003, pp. 271-281 About the author
Conservation-educator Contact: aaa02@fayoum.edu.eg
Abdelrazek Elnaggar is a conservator of organic objects, currently Assistant Lecturer at the Conservation Department of the Faculty of Archaeology at Fayoum University, Egypt. He got a Bachelor’s degree (2000) in Conservation and Restoration of Monuments and Works of Art from the Cairo University and a Master’s degree (2006) in Conservation of Egyptian mummies from Cairo University. He had a scholarship (2007/2008) in laser cleaning of ancient Egyptian Leather at IESL-FORTH in Greece and received a postgraduate diploma in comparative studied on cleaning techniques in relation to Laser cleaning conservation (2008/2009 UK). He has an ongoing PhD thesis focusing on the laser applications to conservation of Egyptian monuments, including collaborative projects in UK with Liverpool University, Natural History Museum, Petrie museum, Imperial College, and UCL, in Italy with Politecnico di Milano, and the Supreme Council of Antiquities (Egypt).
Ahmed Sahab Department of Plant Path., National Research Centre, Dokki, Cairo Siham Ismail Department of Chemistry of Natural Microbial Products, National Research Centre, Dokki Gamal Mahgoub Conservation Department, Faculty of Archaeology, Fayoum University, Alfayoum, Egypt Mohamed Abdelhady Conservation Department, Faculty of Archaeology, Cairo University, Giza, Egypt ▲TOP
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