Microbial Degradation of Ancient Wood Housed in Cheops Boat Museum and Methods of Its Control

The present research paper aims to determine the effect of microbial deterioration on wood housed in Cheops Boat and how to control it. Khufu, the second king of the Fourth Dynasty 2650 BC, established Cheops Boat that was found in 1954 on the southern side of the pyramid. Wood is usually very sensitive to biological attacks. Therefore, fungi, bacteria, and insects can easily attack and metabolize it, leading to signicant physical, chemical, and morphological changes. Cellulose agar was used for the cultivation of fungi, and nutrient agar was used to cultivate bacteria. Microbial infections were identied using light microscopy and PCR after their cultivation. The causative microorganisms were isolated from Cheops Boat and characterized as A. niger, A. avus, A. sulphureus, P. janthinellum, Cladosporium herbarum, Botryotrichum piluliferum, and Bacillus megaterium. The characteristics of the test methods and disinfection include their application to historical objects. Historic wood was analyzed from different perspectives: Stereo microscopes, SEM with EDX, FTIR, as well as wood structure and chemical composition. The biological activities of the isolated microorganisms were studied. The results illustrated that the best concentrations of a specic microbicide for the bio-treatment of the infected wood materials is pentachlorophenol at (900 ppm) as it is sucient to inhibit all isolated microorganisms. It is followed by plant extract of Ceratophyllum demersum at (1000 ppm) concentration, and p-chloro-m-cresol at (2000 ppm) concentration.


Introduction
All historical wooden objects are at risk from insect and fungal attacks (Hunt, 2012) and biological degradation (Walsh et al., 2014). Fungi play a tremendous role in the deterioration of cultural heritage due to their enormous enzymatic activity (Ster inger., 2010). Most of the studies focus on the biodeterioration of valuable wood objects damaged by fungi and bacteria. Furthermore, fungi are very often found in the display and storage conditions of museums (Abdel-Kareem, 2010). In museums and storage rooms, climate control, regular cleaning and microbiological monitoring are essential in order to prevent fungal contamination (Ster inger, 2010) Wood decay by fungi is typically classi ed into three types: soft rot, brown rot, and white rot. The wood decayed by brown rot fungi is typically brown and crumbly. It is degraded by both non-enzymatic and enzymatic systems. White rot fungi are typically associated with hardwood decay, and their wood decay can take different patterns (Goodell et al., 2008). White rotted wood normally has a bleached appearance as a selective decay or a pocket rot. White rot fungi possess both cellulolytic and lignin degrading enzymes. Therefore, they have the potential to degrade the entirety of the wood structure under the correct environmental conditions (Krstulović, et al., 2018). Soft rot fungi typically attack higher moisture, and lower lignin content wood and can create unique cavities in the wood cell wall (Srivastava et al., 2013). The present study aims to choose the best microbicide to stop the growth of all microbial isolates on the wood housed in Cheops Boat.

Sampling
Nine swabs were taken from the Cheops Boat Museum. There were some symptoms of brown spots and spongy wood on the boat wood. Swabs were taken by scratching the surface of the infected materials by sterilized cotton swabs and transferred right onto two prepared agar media (cellulose agar for fungi and nutrient agar for bacteria). Plates were incubated at 28-30°C for 1-21 days, depending on the microorganism.

Media
Media used in this study are Cellulose agar medium and Nutrient agar medium (Atlas, 2005)

Identi cation of microbial isolates
The identi cation of fungal isolates was carried out at the Laboratory of Microbiology, Grand Egyptian Museum, Ministry of Antiquities, whereas the identi cation of bacterial isolates was done by the sequencing of rRNA gene at Solgent Company, South Korea.

Identi cation of fungal isolates
Colonies grown on cellulose agar plates medium were puri ed on the same medium, and every single colony was picked for identi cation according to Gilman (1974) and Domsch et al. (1980a and b) by determining the morphological characteristics using the light microscope (CarlZies Microscope with an analysis unit and a digital camera). They were compared with the standard criteria in references. The identi cation process was performed in the Laboratory of Microbiology, Grand Egyptian Museum, Ministry of Antiquities.

Identi cation of bacteria
Bacterial identi cation was performed based on the molecular approach: Bacteria were grown on nutrient agar at 28° C for 4 days. A small amount of bacterial culture was scraped by sterile spatula suspended in 100µl sterile distilled water in 2ml sterile vials and boiled at 100° C for 15 minutes. Bacterial DNA was extracted and isolated using the SolGent puri cation bead. Before sequencing, the ribosomal rRNA gene was ampli ed using the polymerase chain reaction (PCR) technique in which two universal bacterial primers 27F (forward) and 1492R (reverse) were incorporated in the reaction mixture. Primers used for gene ampli cation have the following composition: The puri ed PCR products (amplicons) were recon rmed using a size nucleotide marker (100 base pairs) by electrophoreses on 1% agarose gel. Then, these bands were eluted and sequenced with the incorporation of dideoxynucleotides (dd NTPs) in the reaction mixture. Each sample was sequenced in the sense and antisense directions using the same primers (Lane, 1991). Sequences were further analyzed using the Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website. Phylogenetic analysis of sequences was done with the help of Meg Align (DNA Star) software version 5.05. moreover, the identi cation of bacterial isolates was done by sequencing the rRNA gene at Solgent Company, South Korea.
2.4. Determination of the activity of the isolated microorganisms a. Enzyme production Production was carried out in 250 ml conical asks. Each contained 100 ml of the production medium (For the production of cellulose enzymes, the main source of carbon (sucrose) was replaced with 10g of cellulose. Flasks were sterilized at 121° C for 15min. After cooling, they were inoculated with 2ml of standard inoculums of each isolate. The inoculated asks were incubated at 28 -30° C for adequate time. At the end of the incubation period, the liquid cultures were centrifuged at 3000 rpm for 15 minutes. The supernatant was taken for the determination of the cellulose enzyme activity, as described below.
b. Enzyme assay Cup plate Clearing Zone Technique (CCZ) was used for assaying the activities of cellulase enzymes. The procedure was carried out by pouring 20 ml aliquots of the detection medium (Ammar et al., 1998) into a sterile Petri dish and was allowed to solidify. A sterile cork borer (15mm diameter) was used to make three cups in each plate, and 0.1 ml of the supernatant (cell-free enzyme) of each isolate was placed into the three cups. Plates were incubated at 30ºC for 24h after which plates were ooded with Lugol's iodine solution to assay cellulase. Enzyme activities were compared based on the diameter (mm) of the clear zone. After that, the isolates showing the highest activity for the enzyme were used in the following experiments.
2.5. Determination of the Minimal Inhibitory Concentration (MIC) of antimicrobial agents against the isolated microorganisms Three commercially available microcides were purchased from Aldrich Company (Germany) and used for testing their effect against the isolated microorganisms and determining their minimal inhibitory concentrations. These microcides were plant extract of Ceratophyllum demersum, Pentachlorophenol, and p-chloro-m-crysol. The mode of action of the antimicrobial agents on the microorganisms was to inhibit active transport by disrupting the cell membranes through solubilizing lipids and denaturing proteins. The cell is more permeable to protons, and ATP synthetase is destroyed (Geis, 2006).
The stock solution of each microcide was prepared by dissolving 1ml/ L ethyl alcohol at 95%. In the case of plant extract gradient, concentrations ranging from 800 to1000 ppm were prepared by diluting the stock solution with alcohol and1g/L ethyl alcohol 95%. Gradient concentrations of other microcodes ranging from 800 to 3000 ppm were prepared by diluting the stock solution with alcohol. One ml of bacterial isolate suspension was spread onto a nutrient agar plate, and one ml of fungi spore suspension (described below) was spread on a cellulose agar plate. Plates were allowed to dry; then, a cork purer(diameter 15 mm) was used to make three pores in each plate. In one plate, 100µl of each concentration (from 800 to 3000 ppm) of the tested microcides were placed in each pore. Plates were incubated

Infection of modern wood with isolated microbial strains
Modern pieces of pine wood specimen were used to determine the effect of isolated microorganisms on their deterioration. The pieces were inoculated with the spore suspension of each speci c isolate by spraying 5 ml of a suspension of each microbial isolate, containing 0.5x10 6 CFU. The infected modern pieces were incubated for 2 months at ambient temperature and 60-70% humidity. Physical and chemical properties of wood pieces were determined before and after inoculation, as described below.
2.6.1. Morphological characteristics Binocular stereomicroscope (in the Laboratory of Microbiology, Grand Egyptian Museum) was used at 8-36X magni cation to examine wood specimens before and after infection.

Chemical properties
The chemical properties of modern wood before and after infection were determined by FTIR and ESEM, as follows: A. Fourier Transform Infrared Spectroscopy (FTIR) Shimadzu-Peristege 21. In IR Lab at the Grand Egyptian Museum was used to identify changes in the shape and linkage of the structure of the tested wood using FTIR apparatus to examine and determine functional groups of compounds according to wavelength and absorbance capacity.

Identi cation of the microbial isolates
The resulted microbial colonies were subjected to preliminary characterization depending on the type of organism.  (Table 1). From these results, it can be seen that the genus Aspergillus was the dominant genus in the nine swabs, followed by Penicillium and Cladosporium. These results are in line with those obtained by (Abed-El Hameed1999) who isolated various species of Aspergillus in addition to Alt. alternate and Cladosporium herbarum from wooden co ns in the Egyptian Museum. Isolates of each genus were subjected to identi cation based on morphological characteristics. The results showed that the isolates of Aspergillus were A.niger, A. avus, and A. sulphureus. The isolates of Penicillium were identi ed as P. janthinellum, and the isolates of other genera were identi ed as Cladosporium herbarum and Botryotrichum piluliferum (Table 1).
Total bacterial counts are shown in Table (2). Bacterial colonies were identi ed by sequencing the 16S rRNA sequencing analysis compared with closely related strains accessed from the Gen Bank (Table 3). The isolates were identi ed as Bacillus megaterium.
Phylogenetic tree based on 16S sequences of rDNA of the bacterial strains isolated in the present study (samples 3, 5, and 7) aligned closely with related sequences and accessed from the GenBank (Figure 2).

Determination of the production by the isolated microorganisms based on the cup plate technique
Data recorded in Table (4) and illustrated in g. (2) indicate that the tested microorganisms varied in their abilities to produce the enzymes. Thus, they vary in the degree of decomposing cellulose. The tabulated data show that the highest cellulolytic activity was observed by Aspergillus avus. These results are in agreement with Pangalloet al., (2007) who found that genus Penicillium and Aspergillus exhibit a relevant cellulolytic activity.

Determination of the Minimal Inhibitory Concentration (MIC) of antimicrobial agents against the isolated microorganisms
To test the effect of antimicrobial agents on the isolated microorganisms, three microcides plant extract of Ceratophyllum demersum, Pentachlorophenol, and p-chloro-m-crysol at concentrations ranging from 800 to 2000 ppm were prepared and applied on the isolated microorganisms. Inhibition zones were measured to determine the MIC that inhibited each species.
The results of the plant extract of Ceratophyllum demersum (Table 5) show that 1000 ppm was the minimum concentration that inhibited all the isolated where the diameter of inhibition zone ranged (19-25 mm). Therefore, it was concluded that using plant extract of Ceratophyllum demersum at a concentration of 1000ppm was the MIC that inhibited all the tested isolates. The results of Penta-cholorophenol showed that at a concentration of 900ppm all isolates were inhibited. Therefore, it can be concluded the MIC of Penta-cholorophenol that inhibited all the tested isolates was 900ppm, and the mean diameter of the inhibition zone ranged 20-42mm.
The results of p-chloro-m-cresol showed that up to 1000ppm, no inhibition was detected. At 2000 ppm, all isolates were inhibited, where the diameter of the inhibition zone ranged (18-21 mm).
From the above results, it can be concluded that pentachlorophenol at 900ppm was the best microcide to stop the growth of all microbial isolates using the lowest possible concentration. These results coincide with Shash and Arya (1999) who recommended spraying pentachlorophenol at 0.25% for protecting objects from fungal biodeterioration and preventing the possible fungal attack.

Effect of microbial infection on physical and chemical properties of modern wood
New pieces of pine wood were infected with the microorganisms isolated from the same material in this study, as follows: Wood pieces were infected with A. niger, A. avus, A. sulphureus, Botryotrichum piluliferum, P. frequentans, and B. megaterium.
Infected pieces were incubated for 2 months at ambient temperature and under 60-70% humidity. The physical and chemical properties of the specimen were determined before and after the infection.

Physical properties of the infected modern wood.
The results presented in Table (6) illustrate changes in the physical properties of wood. They show that black and red spots occurred on the infected pieces of wood. Data also show that the pH of wood decreases as a result of acid production resulting from digesting organic matter by the microbial attack, thereby altering and weakening those materials.
Fungal biodeterioration changes the quality and/or value of material and makes it less functional in utilization terms. A pack of extracellular hydrolytic enzymes excreted by fungi is responsible for the formation of acidic products that Morphological examination obtained by the stereomicroscope showed modern Pinus wood sample ( gure 5a) before infection, and fungal in ltration and mat of lamentous fungal threads on Pinus wood sample(Figures 5b) after infection for 60 days at room temperature. Also, it shows the presence of black and red spots, roughness, and weakness in wood cells. (7) show that no signi cant change was detected in carbon, as well as a decrease in aluminum from 0.59 to 0.18%, and potassium from 0.82 to 0.18%. The following ions appeared after infection: Sodium (1.02%), Silicon (0.30%), Sulfur (0.28%), and Chloride (1.08%). Micrographs and major elements component of modern Pinus wood sample prior infection (Figure 6a, 3. Change in the shape of (C=C) bending linkage movement at a wave number area of (1636-1508 cm-1)

Conclusion
Fungi play a considerable role in the deterioration and degradation of cultural heritage, especially wooden artifacts. In this study, the fungi isolated from the wood housed in Cheops Boat were A. niger, A. avus, A. sulphureus, Botryotrichum piluliferum, and P. frequentans, whereas the bacteria was B. megaterium. The use of plant products will reduce the over-dependence on synthetic chemicals in controlling microbial pathogens and reduce the cost of the management and conservation of archaeological wood. The pentachlorophenol at 900ppm gave good responses and results in controlling the infection (all tested isolates) and showed no changes in the physical, morphological, and mechanical properties of the infected paper. Table (4). Determination of cellulose(s) produced by the isolated microorganisms using the cup plate technique Table 5.Determination of the inhibition zone (mm) of fungal and bacterial species grown on cellulose agar for fungi and nutrient for bacteria as affected three microcides concentration  Determination of cellulose ( s) produced by the isolated microorganisms using the cup plate technique