10.48255/1973-9494.JCSCH.21.2021.05
Dai dipinti murali della tomba egizia di Khety (tomba n. 17, 11 din.) sono stati isolati una specie batterica e cinque funghi. L’attività antimicrobica degli oli essenziali di limone e timo (EO) diluiti in etanolo al 70% è stata valutata rispetto all’isolato batterico (relativo al genere Alcaligenes e alla specie faecalis identificato utilizzando il sequenziamento del gene 16 rRNA) e al fungo predominante nella tomba, Aspergillus niger, Penicillium cyclopium e Fusarium Solani su piastre di agar, utilizzando il metodo della diffusione del disco. Frammenti sono stati prelevati dagli strati della pittura murale per effettuare analisi su repliche (5×5 cm), simili alla pittura murale archeologica, invecchiati artificialmente. L’attività antimicrobica dei due EO studiati è stata valutata rispetto all’isolato batterico e ai tre funghi studiati sulle repliche. È interessante notare che risultati hanno rivelato che l’olio di timo, a una concentrazione del 15%, è generalmente più efficiente del limone contro i tre funghi studiati e, al contrario, l’olio di limone è più efficace contro il batterio rispetto ai funghi. Inoltre le indagini colorimetriche non hanno mostrato cambiamenti significativi nelle repliche dopo il trattamento con i due EO testati con concentrazione del 10% e del 15%. L’analisi GC-MS ha rivelato che il profilo volatile dell’olio di limone è composto principalmente da monoterpeni, con citrale, 6-esoidrossi canfene e Fenchone come composti principali. I risultati hanno mostrato che il timolo (44,38%) è il composto principale per l’olio di timo.
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1973-9494
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Amany M. Reyad
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Conservation Science in Cultural Heritage
analytical technologies
Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings
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2022-07-19
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CSCH
Amany M. Reyad
2021
10.48255/1973-9494.JCSCH.21.2021.05
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Botany Department, Faculty of Science Fayoum University, Egypt
1973-9494
analytical technologies
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Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings
https://conservation-science.unibo.it/article/download/15246/14470
21
E    VALUATING THE ANTIMICROBIAL ACTIVITY
     OF ESSENTIAL OILS IN THE CONSERVATION
     OF MURAL PAINTINGS

   Mayssa D. Albasil
   Conservation Department, Faculty of Archaeology
   Luxor University, Egypt

   Gamal Mahgoub, Abeer ElHagrassy
   Conservation Department, Faculty of Archaeology
   Fayoum University, Egypt

   Amany M. Reyad *     1




   Botany Department, Faculty of Science
   Fayoum University, Egypt

   Keywords: fungi, bacteria, mural painting, essential oils, analytical technologies

   DOI: 10.48255/1973-9494.JCSCH.21.2021.05



   1. Introduction

    Mural paintings are considered as painted history on walls, which provide us with
information about our ancestors; therefore, mural paintings bear essential components
of cultural heritage [1-2]. Unfortunately, due to deterioration factors, these murals can
become discolored and degraded especially as a result of the growth and activity of mi-
crobes [3]. Fungi are recognized as the most effective microorganisms at colonizing
mural paintings [4].
    Mural paintings consist of organic and inorganic materials and are components that
can be used as a nutrient source for microbial colonization [5]. Microbial biodeterioration,
especially fungal deterioration, occurs on mural paintings when conditions such as tem-
perature and relative humidity are favorable to the growth of fungi and spores [2].
    Microbes generally cause aesthetical, physical, and chemical deterioration on differ-
ent mural paintings [6]. Aesthetical deterioration often appears as discoloration on paint-
ings, destroying their aesthetics, stability, and the original intent of beautifying. This dis-
coloration can be caused by the colored pigments (black, green, pink, grey, white, and
purple) produced by some fungi as a result of their metabolic activity.
    Fungi can cause the physical deterioration of mural paintings triggered by the
growth of hyphal networks through the pore space system; the process involves the fil-
amentous structures of the fungi penetrating through the mural painting layers, the
penetration also possibly being favored by turgor pressure inside the hyphae, leading
to cracking, fissures, paint blisters and detachment of the paint layer [7]. Chemical de-
terioration occurs due to microbial metabolic products (including organic and inorgan-



   * Corresponding author: amr01@fayoum.edu.eg
                                                                                                                                                       ic acids and mycotoxins) and extracellular enzymes, which react chemically with mural
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings



                                                                                                                                                       painting components leading to mineral dissolution and structural changes in the com-
                                                                                                                                                       ponents of the mural painting [2].
                                                                                                                                                            Moreover, microbes can release spores, hypha fragments, metabolites, toxins,
                                                                                                                                                       and allergens into the aerosol of the indoor environment of the cultural heritage, af-
                                                                                                                                                       fecting human health at workplaces and causing serious respiratory infections, such
                                                                                                                                                       as bronchial irritation and allergies [8]. Several traditional methods are used for con-
                                                                                                                                                       trolling and treating the fungal deterioration of mural paintings, including mechanical
                                                                                                                                                       methods using brushes, scrapers, and scalpels for the mechanical removal of the
                                                                                                                                                       mycelia, physical methods using UV, IR, γ-Rays, X-rays, etc. which are considered
                                                                                                                                                       short-term treatments and may lead to staining, whitening, or brittleness of historical
                                                                                                                                                       paintings [9]; chemical methods are also employed, using different biocides to inhib-
                                                                                                                                                       it microbial growth on the painted surfaces. However, these biocides have a high tox-
                                                                                                                                                       icological impact on the environment and human health and some of them have been
                                                                                                                                                       banned over time [9]. Unfortunately, these controlling methodologies (traditional,
                                                                                                                                                       physical and chemical) have numerous disadvantages, for instance, toxicity; envi-
                                                                                                                                                       ronmental hazards; higher costs; changing some of the properties of heritage ob-
                                                                                                                                                       jects, etc.
                                                                                                                                                            In this context, natural products could be a favorable alternative to traditional
                                                                                                                                                       methods, in particular, essential oils (EOs) from plants which are recognized as an
                                                                                                                                                       eco-friendly, efficient, low cost, and easy to use approach for the control of various
                                                                                                                                                       types of microorganisms [1]. EOs (also called volatile oils) are aromatic oily liquids ob-
                                                                                                                                                       tained from various plants and extracted from particular parts of the plant, such as
                                                                                                                                                       leaves, stems, flowers, seeds, twigs, bark, wood, fruits, and roots [10]. The antimicro-
                                                                                                                                                       bial activity of EOs is due to their various components including terpenes, aldehydes,
                                                                                                                                                       alcohols, esters, phenolics, ethers, and ketones [10].
                                                                                                                                                            Various studies have been done on the antimicrobial activity of different essential
                                                                                                                                                       oils in the field of cultural heritage conservation, but little is known about their applica-
                                                                                                                                                       tion in the conservation of mural paintings [11]. It is quite difficult to compare the re-
                                                                                                                                                       sults of various studies, as the composition of the essential oils varies greatly depending
                                                                                                                                                       on the kind and age of the plants, the geographical region, and methods of extraction
                                                                                                                                                       of the oil [10].
                                                                                                                                                            This research aims to evaluate the antimicrobial activity of two essential oils (lemon
                                                                                                                                                       oil and thyme oil) on the growth of fungi and bacteria isolated from mural paintings in
                                                                                                                                                       the tomb of Khety (Tomb No. 17) at Beni Hassan, Egypt, and assay the effect of these
                                                                                                                                                       essential oils on the pigments of the mural paintings.


                                                                                                                                                          2. Material and Methods

                                                                                                                                                          2.1. Mural painting case study

                                                                                                                                                           In this research, black biofilms and fragments of the mural paintings in the tomb of
                                                                                                                                                       Khety (Tomb No. 17, 11th Dyn.) were collected (Figure 1). It is one of the Middle King-
                                                                                                                                                       dom rock-cut tombs at Beni Hassan cemetery in Egypt, cut in the cliffs overlooking the
                                                                                                                                                       east bank of the River Nile; its mural paintings were executed using a tempera tech-
                                                                                                                                                       nique. Each of the decorated rock-cut tombs at the site display highly detailed scenes
                                                                                                                                                       of daily life (e.g., agricultural tasks, hunting and fishing, workshop activities, funerary
                                                                                                                                                       rites, etc.).


                                                                                                             126
Figure 1. (A) Mural painting of the Khety tomb (Tomb No. 17, 11th Dyn.) at Beni Hassan, Egypt;
(B) left side of the west wall with visible black biofilms on the chromatic layer.


   2.2. Microbial Isolation and identification

    Fungi on the mural painting were isolated by direct swabbing from the spots show-
ing signs of microbial deterioration; swabs were taken over the four seasons in Janu-
ary, April, August, and November [3]; sterile cotton swabs were wiped over paintings
then transferred to the laboratory in sterile tubes on the same day of collection. For fun-
gal isolation, swabs were cultured directly on previously sterilized yeast extract agar




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
dishes. The isolated fungi were morphologically identified.
    On the nutrient agar dishes, swabs were cultured for bacterial isolation. For the molec-
ular identification of the most predominant bacterial isolate, the genomic DNA was extract-
ed, and the PCR blend was made as described by Hemida and Reyad [12]. The sequenc-
ing of the amplified fragments was completed at GATC Biotech, Constance, Germany.
DNA Sequences were aligned using the NCBI Data Base (www.ncbi.nlm.nlh.gov). The
phylogenetic tree was established using a neighbor-joining technique using TREEVIEW
software (1.6.6) derived from gene sequences of 16SrRNA of some phylogenetic strains
close to the isolated strain.
    All these dishes were sealed with Parafilm and incubated at 28±2°C for five days.
The colonies of fungi that appeared in the dishes after incubation were then purified in
new Petri dishes. A pure culture of each colony type was obtained on each Petri dish.
Yeast extract media (01497, Sigma Aldrich) was used for fungal isolation and purifica-
tion. Based on the samples collected, the predominant three fungal isolates and one
bacterium were chosen to test two essential oils to evaluate their efficacy.


   2.3. Essential oils

   Lemon oil and thyme oil were tested for their antimicrobial activity. The oil extracts
were prepared at Cornell University, New York. Citrus limon (Lisbon variety) leaves were


                                                                                                 127
                                                                                                                                                       used to extract the lemon essential oil. The fresh Citrus leaves were shade-dried at
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings



                                                                                                                                                       room temperature 35 /40°c for 72h and powdered in a blender. 100g of the powder was
                                                                                                                                                       subjected to hydro-distillation for 6h using Clevenger apparatus. The thyme (Thymus
                                                                                                                                                       vulgaris) leaves were collected and dried for five days at room temperature then freeze-
                                                                                                                                                       dried; 100g of the powder was obtained by steam distillation from leaves under natural
                                                                                                                                                       conditions. The two extracted essential oils were kept in dark bottles (Brown bottles) at
                                                                                                                                                       4°C until further investigation. Dimethylformamide (DMF), toluene, and ethanol 70%
                                                                                                                                                       were chosen to dilute the studied EOs to determine the most effective solvent.


                                                                                                                                                          2.4. Mural painting structure

                                                                                                                                                           A small fragment was taken from the mural painting, the support, each pigment and
                                                                                                                                                       the ground layers for analysis and examination.
                                                                                                                                                           X-Ray Diffraction Analysis (XRD)
                                                                                                                                                           An XRD Bruker company model D8 was used for compound identification of the
                                                                                                                                                       samples; it includes reflectometry, featuring high-resolution diffraction, in-plane graz-
                                                                                                                                                       ing incidence diffraction (IP-GID), in addition to small-angle X-ray scattering (SAXS).

                                                                                                                                                          Scanning Electron Microscope (SEM) and EDS
                                                                                                                                                          The examination and analysis of the mural painting samples were carried out by a
                                                                                                                                                       Bruker SEM (Berlin - Germany) equipped with energy-dispersive spectrometry (QUAN-
                                                                                                                                                       TAX EDS) featuring the XFlash® 6 detector series with active areas from 10 to 100 mm2
                                                                                                                                                       combined with the modular ESPRIT software.

                                                                                                                                                           Fourier transmission infrared spectroscopy (FTIR)
                                                                                                                                                           FTIR analyses were executed using a Nicolet Nexus spectrophotometer (Washing-
                                                                                                                                                       ton, USA), coupled with a Nicolet Continuum Fourier transmission infrared spectrosco-
                                                                                                                                                       py microscopy equipped with an HgCdTe detector cooled with liquid N2. Spectra were
                                                                                                                                                       recorded by a Graseby-Specac diamond cell accessory in transmission mode between
                                                                                                                                                       4000 and 700 cm-1.

                                                                                                                                                           Raman spectroscopy
                                                                                                                                                           Raman spectra were recorded using a LabRAM HR Evolution Raman spectrome-
                                                                                                                                                       ter (HORIBA JOBIN YVON) equipped with multi laser capability, a true confocal micro-
                                                                                                                                                       scope, a high-performance Raman spectrometer, multiple detectors and powerful soft-
                                                                                                                                                       ware (LabSpec 6 software).


                                                                                                                                                          2.5. Preparing replica and artificial aging

                                                                                                                                                           In order to evaluate the effectiveness of the essential oils chosen for the study, replicas
                                                                                                                                                       of the mural paintings (executed using a tempera technique) were prepared and aged.
                                                                                                                                                       Ten cycles of thermal aging in an oven at 60ºC were carried out for eight hours followed
                                                                                                                                                       by eight hours at room temperature [13].

                                                                                                                                                          2.6. In vitro antimicrobial assay

                                                                                                                                                           The antifungal activity of EOs was tested using the disc-diffusion method under
                                                                                                                                                       sterile conditions. In this well-known method, agar plates were prepared using Petri


                                                                                                             128
dishes (90 mm diameter), each Petri dish containing 20 ml of autoclaved microbial
agar media (potato dextrose agar for fungal isolation and nutrient agar for bacterial iso-
lation). All plates were inoculated with bacterium (Alcaligenes faecalis) and a spore
suspension of the three tested fungi (Aspergills Niger, Penicillium Cyclopium, Fusari-
um Solani), which was spread over the entire surface of the agar plates by swabbing.
Filter paper discs about 6 mm in diameter (Double Rings® filter paper) were impreg-
nated with the EOs at different concentrations (5%, 10%, and 15%), and 70% ethyl al-
cohol was used for diluting the essential oils, then placed on the agar surface [14].
     Negative control plates were performed by wetting the filter paper discs with 70%
ethyl alcohol. Plates with no oils were used as control plates. The test was performed
in duplicate. All dishes were sealed with Parafilm and incubated at 28±2°C until the mi-
crobial growth fully covered the surface of the medium in the control plates. After incu-
bation, microbial growth was observed and the halo diameter (mm) of inhibition growth
zones around the disc was measured.


   2.6.1. In vitro antimicrobial activity of EOs on replicas

     The most effective concentrations of the essential oils obtained from the above ex-
periment on agar plates were tested on replicas for their antimicrobial activity and
probable effect. Some replicas were treated with EOs at the most effective concentra-
tions of 10% and 15% and 70% ethyl alcohol used for dilution; the other set of replicas
was left untreated as a control. Replicas were inoculated with freshly prepared bacte-
rial suspension (1 x 107 CFU/ml) and spore suspensions (1 x 106 spores/ml) of the
three tested fungi by spreading the suspensions uniformly on the surface under sterile
conditions. All inoculated replicas were put in sealed containers and incubated for two
months and were examined every week.




                                                                                               CONSERVATION SCIENCE IN CULTURAL HERITAGE
   2.7. Colorimetric measurements

    Colorimetric measurements were performed using a portable spectrophotometer,
the Spectro densitometer by eXact X-Rite Switzerland, to measure the different colors
of the replicas before and after being treated with the oils to identify any probable col-
or changes that might have resulted after treatment.


   2.8. Gas chromatography/mass spectrometry (GC/MS) analysis of the EOs

    The GC/MS analysis was performed using a Thermo Scientific, Trace GC Ultra /
ISQ Single Quadrupole MS, TG-5MS fused silica capillary column. For GC/MS detec-
tion, helium was utilized as the carrier gas at a constant flow rate of 1mL/min, and an
electron ionization apparatus with ionization energy of 70 eV was used. The tempera-
ture of the injector and MS transfer line was fixed to 280°C. The oven temperature was
programmed at an initial temperature of 40°C (held for 3 min) to 280°C as a final tem-
perature at an increasing rate of 5°C/min. A percentage of the relative peak area was
used to explore the quantification of all the detected components. The chemicals were
tentatively identified by comparing their respective retention times and mass spectra
with those of the National Institute of Standards and Technology (NIST), WILEY library
database in the GC/MS instrument.


                                                                                             129
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings




                                                                                                                                                       Figure 2. Morphological appearance of the fungi identified in the tomb: (A) Aspergillus niger;
                                                                                                                                                       (B) Aspergillus flavus; (C) Rhizopus stolonifer; (D) Fusarium solani; E) Penicillium cyclopium.



                                                                                                                                                          2.9. Antimicrobial assay of thymol and citral

                                                                                                                                                          Citral (96%) and thymol (98.5%) were purchased from Sigma-Aldrich, and ethanol
                                                                                                                                                       (70%) was used for the preparation of 5, 10, and 15% of the two compounds. The com-
                                                                                                                                                       mon disc-diffusion method was used for antimicrobial detection [14].


                                                                                                              130
    3. Results

    3.1. Microbial identification

    Based on the swabs taken over the four seasons in (January, April, August and No-
vember), the data shows that deterioration of the tomb was caused by the effects of
five fungi: Aspergillus niger, Aspergillus flavus, Fusarium solani, Rhizopus stolonifer,
Penicillium cyclopium) (Figure 2). It can be seen clearly from molecular identification
that the tested bacterium was included in the genus Alcaligenes and closely related to
the species faecalis. It showed the highest sequence similarities with the Alcaligenes
faecalis strain NBRC 13111 (Figure 3).




                                                                                                     CONSERVATION SCIENCE IN CULTURAL HERITAGE

Figure 3. Phylogenetic dendrogram obtained by the distance matrix analysis of 16S rRNA se-
quences, showing the position of the isolated bacterium strain among phylogenetic neighbors.
The dark circle indicates the position of our isolated strain.


    3.2. Dilution of the essential oils

    The tested essential oils were diluted with dimethylformamide (DMF); they mixed
very well and formed a homogeneous mixture. However, it was noticed that dimethyl-
formamide affected the color of the tested EOs, as the color of the liquids became
darker in color and over time the color change increased (Figure 4).
    When the tested EOs were diluted with toluene, they did not mix and form a heteroge-
neous mixture (Figure 5). By diluting in ethanol 70%, it was noticed that ethanol 70% did
not affect the color of the tested oils, and it mixed very well with the oils in the first hour,
but after a few hours, a thin layer of the oils appeared on the surface (Figures 6 and 7).


                                                                                                   131
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings




                                                                                                                                                       Figure 4. Color change of thyme EO when diluted with DMF. A) Directly after dilution; B) after 7 days.




                                                                                                                                                                                                        Figure 6. EOs directly after diluting with ethanol
                                                                                                                                                       Figure 5. Thyme EO diluted with Toluene.         70%. A) Thyme; B) lemon.




                                                                                                                                                                Figure 7. EOs diluted with ethanol 70% after a few hours. A) Thyme; B) lemon


                                                                                                                    132
   3.3. Analysis of Mural painting structure (support, pigments and media)

   3.3.1. X-Ray Diffraction Analysis (XRD)

    As shown in Figure 8, the XRD analysis of the support showed 83.9% calcite
(CaCO3) and 16.1% magnesium calcite (CaMg(CO3)2).
    For the ground layer, the XRD analysis indicated 69% calcite (CaCO3), 30% anhy-
drite (CaSO4), and 1% quartz (SiO2).
    XRD analysis of the painted layer showed that the red pigment sample consists of
(96%) calcite (CaCO3), 2.8% quartz (SiO2), and 1.2% hematite (Fe2O3); the yellow pig-
ment sample consists of 61.1% goethite (FeO(OH)), 2.30% calcite (CaCO3), 5.2% an-
hydrite (CaSO4), and 5.3% quartz (SiO2).




                                                                                          CONSERVATION SCIENCE IN CULTURAL HERITAGE




                   Figure 8. XRD pattern of the mural painting samples.


                                                                                        133
                                                                                                                                                           The ground layer results showed 51.2% calcite (CaCO3), 28.3% vaterite (CaCO3),
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings



                                                                                                                                                       18.8% cuprorivaite (CaCuSi4O10) the basic constituent of Egyptian blue, and 1.6%
                                                                                                                                                       quartz (SiO2) for the blue pigment sample. XRD analysis of the green pigment sam-
                                                                                                                                                       ple showed that it consists of 35.9% calcite (CaCO3), 32.7 malachite (Cu2CO3(OH)2),
                                                                                                                                                       20.1% cristobalite (SiO2), and 11.3% goethite (FeO(OH)) and showed 23.8% anhy-
                                                                                                                                                       drite (CaSO4), 23.6% calcite (CaCO3), 22.7% nitratine (NaO3), 13.3% cristobalite
                                                                                                                                                       (SiO2), 9.1% calcium sulphate (CaSO4), 5.4% calcium hydroxide (Ca(OH)2), and 2.2%
                                                                                                                                                       halite (NaCl) for the white pigment. XRD analysis of the black pigment sample
                                                                                                                                                       showed that it consists of (38.8%) calcite (CaCO3), 27.6% Dolomite (CaMg(CO3)2),
                                                                                                                                                       12.2% carbon (C), 8.9% calcium carbide (CaC2), 7% perkovaite, 3.5% lawrencite
                                                                                                                                                       and 1.9% cohenite.


                                                                                                                                                          3.3.2. Scanning Electron microscope (SEM) and EDS

                                                                                                                                                           The analysis showed that the support consists of Ca, C, O the elements of calcite
                                                                                                                                                       (CaCO3), and Ca, Mg, C, O the elements of dolomite (CaMg (CO3)2). The analysis
                                                                                                                                                       showed that the ground layer consists of Ca, C, O, the elements of calcite (CaCO3);
                                                                                                                                                       Si, O, the elements of quartz (SiO2); and Ca, S, O, the elements of anhydrite (CaSO4).
                                                                                                                                                       For the painted layer as shown in Figure 9, the red pigment sample analysis showed




                                                                                                                                                                          Figure 9. EDS-SEM pattern analysis of the pigments.


                                                                                                                134
the presence of O, Fe, the basic constituents of hematite (Fe2O3) and the yellow pig-
ment sample analysis showed the presence of O, Fe, the basic constituents of goethite
(FeO(OH)). The blue pigment sample analysis showed the presence of O, C, Ca, Si,
Cu, the basic constituents of Egyptian blue (CaCuSi4O10), the green pigment sam-
ple analysis showed the presence of O, C, Cu, the basic constituents of malachite
(Cu2CO3(OH)2), the white pigment sample analysis showed the presence of O, C, Ca,
the basic constituents of calcite (CaCO3), and the black pigment sample analysis showed
the presence of a large amount of C and traces of other elements (Ca, Si, Mg, Cu, S, Al)
indicated the use of charcoal as a source of the black pigment. SEM examination of the
archeological pigment samples showed the presence of fungal hyphae between grains
(Figure 10).




                                                                                                 CONSERVATION SCIENCE IN CULTURAL HERITAGE




Figure 10. SEM micro-image showing the penetration of fungal mycelium between mural painting
grains.


     A pigment fragment was analyzed by FTIR to identify the binding medium that was
used in the painting process. FTIR spectra showed that Arabic Gum was used as a
binding medium. The sample was characterized in the presence of the activated group
as: (O-H) in 3423.99, (C-H) in 2975.62, (O-H) in 1624.73, (C-H) in 1424.17 and (C-O)
in 874.56 (Figure 11). Another sample was analyzed by Raman spectroscopy to iden-
tify the medium used in the painting process. Raman spectra showed that Arabic Gum
was used as a binding medium. The sample was characterized in the presence of the
activated groups for Arabic Gum.


                                                                                               135
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings




                                                                                                                                                       Figure 11. Arabic Gum is used as a binding medium of the pigments. a) FTIR pattern; b) Raman
                                                                                                                                                       spectra.



                                                                                                                                                           3.4. In vitro antimicrobial assay

                                                                                                                                                           The results of the antimicrobial activity of the two studied EOs against Aspergillus
                                                                                                                                                       niger, Penicillium cyclopium, Fusarium Solani and Alcaligenes faecalis on agar plates,
                                                                                                                                                       revealed that the two oils had an inhibitory effect on the growth of studied microbes,
                                                                                                                                                       and that an increase in oil concentration leads to an increase of inhibition zones (Table 1).
                                                                                                                                                       The inhibitory action results in the appearance of a transparent halo (inhibition growth
                                                                                                                                                       zone) around the filter paper disc impregnated with the studied essential oils.
                                                                                                                                                           Results revealed that thyme oil has a more powerful antifungal effect than antibac-
                                                                                                                                                       terial activity. It was observed that 5% thyme oil has neither an antifungal effect against
                                                                                                                                                       Fusarium Solani nor against Alcaligenes faecalis bacterium, while the average diameter

                                                                                                                                                       Table 1. Average of the Inhibition Zone diameter for the two tested EOs against four microbes; (√)
                                                                                                                                                       completely inhibited microbial growth; (0) no microbial effect.




                                                                                                                136
of the inhibition growth zone was 8mm for Aspergillus niger and 11 mm for Penicillium
cyclopium. Thyme oil at 10% concentration completely inhibited the mycelia growth of
Fusarium Solani, while the average diameter of the inhibition growth zone was 11 mm
for Aspergillus niger, 14 mm for Penicillium cyclopium, and 7 mm for Alcaligenes faeca-
lis. Thyme oil at 15% concentration completely inhibited the mycelia growth of Penicilli-
um cyclopium and Fusarium Solani, while the average diameter of the inhibition growth
zone of Aspergillus niger was 24 mm and 9 mm for the bacterium.
     The powerful antibacterial activity of lemon oil in different concentrations was detect-
ed where all concentrations (5, 10, and 15 %) inhibited the growth of Alcaligenes faeca-
lis completely. Lemon oil at 5% concentration has no antifungal effect against all the fun-
gal strains, as there is no inhibition growth zone around the filter paper discs. Lemon oil
at 10% concentration completely inhibited the mycelia growth of Fusarium Solani, while
the average diameter of the inhibition growth zone was 8mm for Aspergillus niger and
9mm for Penicillium cyclopium. Lemon oil at 15% concentration completely inhibited the
mycelia growth of Penicillium cyclopium and Fusarium Solani, while the average diame-
ter of the inhibition growth zone of Aspergillus niger was 14mm. Ethanol 70% (as a neg-
ative control) has no antimicrobial effect against the four studied microbes.


    3.5. Assessment for in vitro antimicrobial activity of EOs on replicas

    Figures 12 and 13 illustrate the impact of antimicrobial activity of the two studied EOs
oils against Aspergillus niger, Penicillium cyclopium, Fusarium Solani, and Alcaligenes




                                                                                                         CONSERVATION SCIENCE IN CULTURAL HERITAGE




Figure 12. Antimicrobial activity of thyme EO against 1: Fusarium Solani + bacteriumon on replicas.
2: Penicillium cyclopium + bacterium on replicas. 3: Aspergillus niger + bacterium. (A) Control; (B)
thyme EO 10 %; (C) thyme EO 15%.


                                                                                                       137
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings




                                                                                                                                                       Figure 13. Antimicrobial activity of lemon EO against 1: Fusarium Solani + bacteriumon on replicas.
                                                                                                                                                       2: Penicillium cyclopium + bacterium on replicas. 3: Aspergillus niger + bacterium. (A) Control;
                                                                                                                                                       (B) lemon EO 10 % ;(C) lemon EO 15%.


                                                                                                                                                       faecalis on agar plates proving the efficacy of the two essential oils at concentrations of
                                                                                                                                                       10% and 15%, so the two EOs at concentrations of 10% and 15% were applied to the
                                                                                                                                                       replicas with the bacterium and the three studied fungi. After an incubation period of
                                                                                                                                                       three months (considering they were examined every week) and by visual observation
                                                                                                                                                       of the control replicas (only inoculated with bacterial and spore suspensions of the three
                                                                                                                                                       tested fungi), it was found that a notable growth of fungi occurred on the surface of the
                                                                                                                                                       replicas. The results of the visual examination of the other replicas (treated with EOs at
                                                                                                                                                       different concentrations and inoculated with spore suspensions) revealed that the high-
                                                                                                                                                       er the oil concentration, the more efficient it is in inhibiting fungal growth; thyme oil is
                                                                                                                                                       generally more efficient than lemon oil against the three studied fungi, thyme oil at 10%
                                                                                                                                                       and 15% concentrations inhibited the growth of the three studied fungi on all the repli-
                                                                                                                                                       cas (no fungal growth appeared on the surface of the replicas compared to the control
                                                                                                                                                       replicas), while lemon oil inhibited the growth of the three studied fungi only at 15% con-
                                                                                                                                                       centration, while the 10% concentration only inhibited the growth of Fusarium solani.
                                                                                                                                                            By swabbing the different replicas to monitor bacterial abundance, it was noticed
                                                                                                                                                       that the concentrations of lemon oil at 10 and 15 % have a powerful activity against Al-
                                                                                                                                                       caligenes faecalis, as there is no visible growth by culturing the swabs on agar plates.


                                                                                                                                                           3.6. Colorimetric measurements

                                                                                                                                                           The obtained results of the colorimetric measurements of all pigments of the aged
                                                                                                                                                       replicas treated with EOs at 10% and 15% concentrations were measured using a


                                                                                                                138
spectrophotometer (the CIE L*,a*,b* color system was used). Total color change (ΔE)
was calculated according to the following equation:

                           ΔE* = [(ΔL*)2+ (Δa*)2 + (Δb*)2]1/2

    The results in Figure 14 show that no notable changes occurred due to the effect
of the two EOs either in the concentration of 10% or 15%.




                                                                                                CONSERVATION SCIENCE IN CULTURAL HERITAGE


Figure 14. Change in color before and after treatment by EOs at 10% and 15% concentrations.



   3.7. Chemical composition of EOs

     The GC/MS analysis of the lemon oil showed a total of 49 volatile organic com-
pounds, most of which are terpenoids; they were grouped into homologous series as
follows: monoterpene hydrocarbons, oxygenated monoterpenes, oxygenated sesquit-
erpenes and sesquiterpenes, diterpenes. Table 2 shows that 17 compounds were clas-
sified as monoterpenes, with citral (15.34%), camphene, and 6-exohydroxy camphene
(10.12%), and fenchone (7.14%) as the major compounds. Six compounds were listed
as sesquiterpenes with funebrene (2.11%) as the major compound and only one com-
pound that was listed as a diterpene, cembrene (0.55 %).


                                                                                              139
                                                                                                                                                       Table 2. Chemical composition of lemon oil analyzed by gas chromatography–mass spectrometry
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings



                                                                                                                                                       (GC-MS).

                                                                                                                                                        Monoterpene hydrocarbons                            RT(min)   Peak area (%)   M.wt   M. formula
                                                                                                                                                        α-Pinene                                            7.57      0.13            136    C10H16
                                                                                                                                                        Camphene                                            7.65      0.57            136    C10H16
                                                                                                                                                        Delta 3-Carene                                      8.11      0.26            136    C10H16
                                                                                                                                                        α-Myrcene                                           9.93      5.13            136    C10H16
                                                                                                                                                        α-Terpinene                                         11.73     0.47            136    C10H16
                                                                                                                                                        γ-Terpinene                                         12.02     1.27            136    C10H16
                                                                                                                                                        α-Myrcene                                           22.73     0.11            136    C10H16
                                                                                                                                                        Oxygenated monoterpenes                             RT(min)   Peak area (%)   M.wt   M.formula
                                                                                                                                                        Eucalyptol                                          11.17     1.03            154    C10H18O
                                                                                                                                                        Linalool oxide                                      12.46     0.16            170    C10H18O2
                                                                                                                                                        6,6-dimethyl(CAS) beta-pinene oxide                 13.12     0.18            152    C10H16O
                                                                                                                                                        Fenchone                                            13.39     7.14            152    C10H16O
                                                                                                                                                        Pulegone                                            14.43     0.86            152    C10H16O
                                                                                                                                                        α-Cyclocitral                                       15.35     3.09            152    C10H16O
                                                                                                                                                        Linalool                                            15.62     0.67            154    C10H16O
                                                                                                                                                        6-exohydroxy camphene                               18.75     9.37            152    C10H16O
                                                                                                                                                        Citral                                              20.13     15.34           152    C10H16O
                                                                                                                                                        Sesquiterpenes                                      RT(min)   Peak area (%)   M.wt   M.formula
                                                                                                                                                        α-Funebrene                                         33.62     2.11            204    C15H24
                                                                                                                                                        Oxygenated Sesquiterpenes                           RT(min)   Peak area (%)   M.wt   M.formula
                                                                                                                                                        Elemol                                              26.89     0.12            222    C15H26O
                                                                                                                                                        α-Cadinol                                           27.36     0.28            222    C15H26O
                                                                                                                                                        Farnesol (1st isomer)                               32.46     0.11            222    C15H26O
                                                                                                                                                        Farnesol (2 isomer)
                                                                                                                                                                     nd
                                                                                                                                                                                                            35.79     0.19            222    C15H26O
                                                                                                                                                        Trans-Farnesol (3rd isomer)                         36.41     0.21            222    C15H26O
                                                                                                                                                        Diterpenes                                          RT(min)   Peak area (%)   M.wt   M.formula
                                                                                                                                                        Cembrene                                            34.22     0.55            272    C20H32
                                                                                                                                                        Alkanes/alkenes, Aldhydes/ketones,        Esters,   RT(min)   Peak area (%)   M.wt   M.formula
                                                                                                                                                        ethers, and Orgnic acids
                                                                                                                                                        4-Cyclopropyl-4-penten1-ol                          5.13      0.36            124    C8H12O
                                                                                                                                                        Salvene                                             6.86      0.12            124    C9H16
                                                                                                                                                        7,7-dichloro-6-[(methylsulfonyl)oxy-bicyclo-hept-   7.26      0.27            256    C8H4D6Cl2O3S
                                                                                                                                                        2-ene
                                                                                                                                                        7-Methylene-9-oxa-bicyclo-non-2-ene                 9.49      1.47            136    C9H12O
                                                                                                                                                        6-Methyl-5-hepten-2-one                             10.22     12.15           126    C8H14O
                                                                                                                                                        3,7,7-Trimethyl-1,3,5-cycloheptatri-ene             11.01     1.67            134    C8H14
                                                                                                                                                        2-methyl-2-hepten-5-yn4-one                         12.89     0.36            122    C8H10O
                                                                                                                                                        Fenchyl acetate                                     13.97     0.23            196    C12H20O2



                                                                                                       140
 (9-Oxabicyclo-non-6-en-3-yl) methanol             14.74     4.77             154    C9H14O2
 4,7,7-trimethylbicyclo-hept-4-ene-3-ol            15.98     1.12             152    C10H16O
 3,5-Diacetoxy-2-methylcyclohexanone               16.25     0.11             228    C11H16O5
 2-Isopropenyl-1-methylcyclo-butane ethanol        17.39     0.42             154    C10H18O
 3-cyano-5-hydroxy-2,5-dimethyl-4-oxy-pyrrole      18.81     8.52             152    C7H8N2O2
 2-methoxy4,4,6-trimethyLcyclohexa-2,5-dien-1-     19.63     4.36             166    C10H14O2
 one
 1-[1-(trimethylsilyl)methyl-ethenyl]              19.73     3.42             198    C11H22OSi
 cyclo-pentanol
 1,5,5-Trimethyl-6-methylene-cyclohexene           21.78     8.91             136    C10H16
 1,4,4-Trimethylcyclohex-2-enyl)acetic acid        22.3      0.12             182    C11H18O2
 2-Oxo-1,2-dihydro-1-azaanthraquinone              23.29     0.7              225    C13H7NO3
 1-Heptatriacotanol                                35.28     0.14             536    C37H76O
 Hexadeca-2,6,10,14-tetraen-1-ol                   35.96     0.15             290    C20H34O
 Farnesol methyl ether                             36.15     0.12             236    C16H28O
 Methyl-5-oxo-2-phenyl-3-propylteTrahydrofuran-    36.49     0.19             262    C15H18O4
 2-carboxylate
 Oxiranecarboxaldehyde3-methyl-3- (4-methyl-3-     37        0.3              168    C10H16O2
 pentenyl)
 4-Propyl-3-oxabicyclo-decane                      37.14     0.15             182    C12H22O
 Linalyl isobutyrate                               37.45     0.12             224    C14H24O2

RT: retention time for each compound (minutes), peak area % represents the concentration of
each compound in the oil sample, M. wt: molecular weight of the compound, and M. formula: mo-
lecular formula




                                                                                                    CONSERVATION SCIENCE IN CULTURAL HERITAGE
    Forty-six compounds for the thyme oil were reported, six compounds of monoter-
pene hydrocarbons were detected, of pinene (4.4%) and Camphene (2.03%), oxygen-
ated monoterpenes of thymol (44.58%), linalool (3.28%) and cineole (1.64%), six com-
pounds of sesquiterpenes were listed, and many other organic compounds that were
classified as alcohols, esters, acids, and others (Table 3).


Table 3. Chemical composition of thyme oil analyzed by gas chromatography–mass spectrometry
(GC-MS).

 Monoterpene hydrocarbons                       RT(min)     Peak area (%)     M.wt   M. formula
 α-Thujene                                      7.88        0.46              136    C10H16
 α-Pinene                                       8.03/8.12   4.40              136    C10H16
 Camphene                                       8.55        2.03              136    C10H16
 α-Terpinene                                    9.42        0.82              136    C10H16
 Bornylene                                      10.47       0.58              136    C10H16
 γ-Terpinene                                    12.13       0.60              136    C10H16
 Oxygenated monoterpenes                        RT(min)     Peak area   (%)   M.wt   M.formula
 1,8-Cineole                                    11.48       1.64              154    C10H18O



                                                                                                  141
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings


                                                                                                                                                       Borneol                                        15.70         0.68              154    C10H18O
                                                                                                                                                       L-Linalool                                     13. 70        3.28              154    C10H18O
                                                                                                                                                       Thymol                                         19.88         44.58             150    C10H14O
                                                                                                                                                       Sesquiterpenes                                 RT(min)       Peak area   (%)   M.wt   M.formula
                                                                                                                                                       α-Humulene                                     10.65/22.95   0.34/0.11         204    C15H24

                                                                                                                                                       α-farnesene                                    22.49         0.09              204    C15H24
                                                                                                                                                       γ-Cadinene                                     23.50         0.19              204    C15H24
                                                                                                                                                       Germacrene                                     24.41         0.19              204    C15H24
                                                                                                                                                       Cadinene                                       24.63         0.31              204    C15H24
                                                                                                                                                       α-Muurolene                                    27.31         0.28              204    C15H24
                                                                                                                                                       Oxygenated Sesquiterpenes                      RT(min)       Peak area   (%)   M.wt   M.formula
                                                                                                                                                       Caryophyllenyl alcohol                         25.71         0.08              222    C15H26O
                                                                                                                                                       Alkanes/alkenes, Aldhydes/ketones, Esters,     RT(min)       Peak area   (%)   M.wt   M.formula
                                                                                                                                                       ethers, and Orgnic acids
                                                                                                                                                       Propanoic acid 2-methyl-tri-ethylsilyl ester   5.59          0.13              160    C7H16O2Si
                                                                                                                                                       Krypton                                        6.62          0.10              84     Kr
                                                                                                                                                       2-Phenylacetic acid                            7.96          1.40              136    C8H8O2
                                                                                                                                                       2-Decen-1-ol                                   9.70          0.61              156    C10H20O
                                                                                                                                                       Geranyl propionate                             10.02         3.29              210    C13H22O2
                                                                                                                                                       1-(-1Hydroxy1methylethyl)-cyclohexanol         10.49         0.50              158    C9H18O2
                                                                                                                                                       Benzenamine-,2,4,6-trimethyl                   10.95         1.24              135    C9H13N
                                                                                                                                                       Dicinnamyl Ether                               11.13         0.61              250    C18H18O
                                                                                                                                                       1-(Acetylmethyl)1-vinyl Cyclo-pentane          14.68         0.62              152    C10H16O
                                                                                                                                                       4-Fluoro-4-isopropylcyclo                      14.94         0.44              154    C8H7FO2
                                                                                                                                                       Hexa-2,5-dienone
                                                                                                                                                       Endobornyl acetate                             15.89         1.44              196    C12H20O2
                                                                                                                                                       3-Cyclohexen-1-ol-4-methyl-1-                  15.96         0.27              154    C10H18O
                                                                                                                                                       (1-methylethyl)
                                                                                                                                                       3,7-Dimethylocta-2,6-dienyl 2-acetylbenzoate   16.39         1.16              300    C19H24O3
                                                                                                                                                       1,3-Cyclohexadiene-1-methanol-                 16.51         3.02              166    C11H18O
                                                                                                                                                       1,2,6,6-tetramethyl

                                                                                                                                                       Methyl-4-(aminomethyl) benzoate                17.33         0.48              165    C9H11NO2
                                                                                                                                                       Methyl thymyl ether                            17.57         0.30              164    C11H16O
                                                                                                                                                       Estragole                                      18.89         2.15              148    C10H12O
                                                                                                                                                       4-nitrobenzoate                                19.26         0.11              291    C14H10ClNO4
                                                                                                                                                       2H(1)-4-MethoxyPhenyl-ethene                   19.99         4.48              134    C9H9DO
                                                                                                                                                       Methyl Cinnamate                               21.51         0.14              162    C10H10O2
                                                                                                                                                       Methyleugenol                                  22.01         0.19              178    C11H14O2
                                                                                                                                                       Benzyl (2-Methylpent-4-enyl) amine             22.24         2.54              189    C13H19N
                                                                                                                                                       Oxirane, (hexadecyloxy)methyl                  31.37         0.16              298    C19H38O2

                                                                                                                                                       2,5-Dimethyl-para-nisaldehyde                  31.51         0.63              164    C10H12O2



                                                                                                             142
 2,5-dimethyl anisole                      32.62        0.26             136       C9H12O
 2,4,5,6-Tetramethylpyrimidine             32.91        0.08             136       C8H12N2
 N-Cyanomethyl-2-azidobenzamide            34.03        0.09             201       C 9H 7N 5O
 3-(4-Methyl-3-pentenyl)-3-cyclo-hexenyl   34.42        0.20             262       C18H30O
 pentylketone
 Hexadecanoic acid,tri-methylsilyl ester   35.20        0.54             328       C19H40O2Si


RT: retention time for each compound (minutes), peak area % represents the concentration of
each compound in the oil sample, M. wt: molecular weight of the compound, and M. formula: mo-
lecular formula



    3.8. Antimicrobial assay of thymol and citral

    The antimicrobial activity results of 5, 10, and 15% of the two compounds are con-
sistent with those obtained from the two EOs with slight changes (Table 4). For thymol,
complete inhibition was observed only for Penicillium cyclopium at 10 and 15%. Citral
at 15% concentration completely inhibited Alcaligenes faecalis, while the average di-
ameter of the inhibition growth zones were 10mm, 11mm, and 12mm for Fusarium So-
lani, Penicillium cyclopium, and Aspergillus niger respectively. At 10% of citral, inhibi-
tion zones were detected only for Aspergillus niger and Alcaligenes faecalis. Thymol
and citral at 5% concentration had no antifungal effect (there is no inhibition growth
zone around the filter paper discs). Ethanol 70% (as a negative control) had no antimi-
crobial effect against the four studied microbes.

Table 4. Average diameter of the Inhibition Zone for the thymol and citral against four microbes,
(√) completely inhibited microbial growth, (0) no microbial effect.




                                                                                                      CONSERVATION SCIENCE IN CULTURAL HERITAGE




Data are the average of three replicates ± standard errors


                                                                                                    143
                                                                                                                                                          4. Discussion
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings




                                                                                                                                                            In this study, the most predominant bacterium and three fungal species were isolat-
                                                                                                                                                       ed from the mural paintings of the Egyptian tomb of Khety (Tomb No. 17, 11th Dyn.).
                                                                                                                                                       The mural paintings of the Khety tomb were executed using a tempra technique which
                                                                                                                                                       is the main technique used in ancient Egypt [15]. Based on the swabs taken over the
                                                                                                                                                       four seasons, the data indicates that the deterioration of the tomb was caused by the
                                                                                                                                                       four predominant microbes Aspergillus niger, Penicillium cyclopium, Fusarium Solani,
                                                                                                                                                       and Alcaligenes faecalis.
                                                                                                                                                            The results obtained regarding the antifungal activity of the two studied EOs (lemon
                                                                                                                                                       and thyme oils) against Aspergillus niger, Penicillium cyclopium, Fusarium Solani, and
                                                                                                                                                       Alcaligenes faecalis (the most common and dominant microbes in the tomb) on agar
                                                                                                                                                       plates and replicas, revealed that all the studied EOs have variant inhibitory microbial ef-
                                                                                                                                                       fects and thyme oil at 15% concentration is generally more efficient than lemon oil against
                                                                                                                                                       the three studied fungi at the same concentration. Some previous studies have also re-
                                                                                                                                                       ported that lemon and thyme EOs have antifungal activity against many fungi isolated
                                                                                                                                                       from archaeological objects [3].
                                                                                                                                                            Dimic et al. [16] tested the antifungal activity of lemon EO and coriander, and cin-
                                                                                                                                                       namon extracts, by direct contact and vapour phase, indicating that the lemon essen-
                                                                                                                                                       tial oil showed complete inhibition of the growth of the tested microbes, while the cin-
                                                                                                                                                       namon extract (in tested concentrations: 1.25-5.83 μl/ml) was the weakest inhibitor for
                                                                                                                                                       the tested fungi.
                                                                                                                                                            The main difficulty in the dilution process of essential oils is their low water solubil-
                                                                                                                                                       ity, so the addition of solvents or detergents is unavoidable [17]. Most researchers
                                                                                                                                                       have used ethanol or dimethyl sulfoxide (DMSO) to dilute essential oils [18,19]. Other
                                                                                                                                                       researchers have used dimethylformamide, chloroform, and ethanol 70% to dilute es-
                                                                                                                                                       sential oils; however, due to the damage/harm caused by dimethylsulfoxide (DMSO)
                                                                                                                                                       and chloroform to archaeological materials and human health, they were excluded.
                                                                                                                                                       The effectiveness of dimethylformamide (DMF), toluene, and ethanol 70% to dilute the
                                                                                                                                                       studied EOs was first tested to determine the most effective solvent. The test revealed
                                                                                                                                                       that ethanol 70% was the most effective solvent to dilute the studied EOs; it did not af-
                                                                                                                                                       fect the color of the two tested EOs; it mixed very well with the oils; and when tested
                                                                                                                                                       as a negative control it showed no antimicrobial effect against the three studied fungi.
                                                                                                                                                       This result agreed with the results found by Gatti et al. [14].
                                                                                                                                                            The analysis of the volatile profile of the lemon oil shows that it mainly contains
                                                                                                                                                       monoterpene, with citral, 6-exohydroxy camphene, and fenchone as the major com-
                                                                                                                                                       pounds. These results conflict with many of the previously mentioned results [10]
                                                                                                                                                       where limonene was the main constituent, with values of 78.93 and 53.57%, followed
                                                                                                                                                       by b-pinene (5.08 and 7.44%) and supported by those of Tao et al. [20], who found
                                                                                                                                                       that monoterpene hydrocarbons (C10H16) were the majority (88.96 percent, w/w) in
                                                                                                                                                       Citrus reticulata Blanco essential oil, with citral, citronellol, octanal, linalool, decanal,
                                                                                                                                                       nonanal, b-pinene, and c-terpinene as the major compounds. In our investigation, thy-
                                                                                                                                                       mol was detected as the major compound in the thyme oil and this result is supported
                                                                                                                                                       by those obtained by Shabnum and Wagay [21], who demonstrated that thymol and
                                                                                                                                                       γ-terpinene were the main constituents of thyme oil. Satyal et al [22] also studied the
                                                                                                                                                       composition of T. vulgaris oil from different regions, the oil sample from Jablanicki,
                                                                                                                                                       Serbia, was of the geraniol chemotype (geraniol, 59.8%; geranyl acetate, 16.7%); the oil
                                                                                                                                                       from Nyons, France, was of the linalool chemotype (linalool, 76.2%; linalyl acetate, 14.3%);
                                                                                                                                                       and the essential oil from Richerenches, France, was of the thymol chemotype


                                                                                                         144
(thymol, 47.1%; p-cymene, 20.1%), and the sample from Pomoravje District, Serbia,
was of the sabinene hydrate chemotype (cis-sabinenehydrate, 30.8%; trans-sabinene
hydrate, 5.0%).
    It is quite difficult to compare the results obtained from different studies, due to the
fact that the composition of the EOs can vary greatly depending on the geographical
species and age of the plants, and methods of drying and extracting the oil. The mech-
anisms of the antimicrobial activity of EOs are not fully understood. Therefore, a pleth-
ora of studies suggest that this antifungal activity may be due to degrading of the cell
wall, preventing the fungus to sporulate or the conidia to germinate, the flowing of cel-
lular content, inactivating of extracellular enzymes, and thickening of the cytoplasm
[23]. The antimicrobial activity of citral and thymol, and their concordance with the oils,
suggest that the oil-forming compounds have a synergistic antimicrobial effect, which
indicates that these compounds work together to eradicate microorganisms.


      5. Conclusion

     Lemon and thyme EOs diluted in ethanol 70% have antimicrobial activity against
the most predominant microbes isolated from the mural paintings in the Egyptian
tomb of Khety, both on agar plates and replicas. Results obtained for the two oils
were slightly different, as thyme EO at 15% concentration was more effective than
lemon oil against the three studied fungi. As regards antibacterial activity, lemon oil
was the most effective. Colorimetric measurements showed that no notable changes
occurred due to the treatment with the two tested EOs. Significant antimicrobial ac-
tivity was observed using citral and thymol which were the major detectable com-
pounds in the EOs.


      References




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   Biographical notes

    Amany M. Reyad is currently a lecturer of microbiology at the Botany Department,
Faculty of Science, Fayoum University. She received her doctorate degree in microbi-
ology from Fayoum University in 2018. She has published several papers discussing
different issues related to environmental and applied microbiology. She has taught
many courses on the Microbiology Diploma course to students in the Faculty of Sci-
ence, Fayoum, Egypt.

    Mayssa Dayhoum Albasil is a lecturer in the conservation department at Luxor
University, Egypt. She got her Ph.D degree in August 2021 in applied Microbiology in
the conservation of mural painting from Fayoum University, Egypt. She got her Master
degree in conservation of mural painting in 2015 from Cairo University, Egypt.

    Gamal Mahgob is a professor in conservation of organic and inorganic materials
in cultural heritage at Fayoum University, Egypt. He is a Vice Dean of the Faculty of Ar-
cheology, Fayoum University for education and students. He was formerly the Head of
the Central Department of Conservation and Restoration at the Ministry of Antiquities.
Professor Gamal Mahgoub is the supervisor of more than 100 master students and 93
Ph.D. students. He is an Editor for Scopus’ Shedet journal and a reviewer for 19 inter-
national journals. His main research interests are in applied nanotechnology in cultur-




                                                                                               CONSERVATION SCIENCE IN CULTURAL HERITAGE
al heritage, laser technology in the conservation of culture heritage, and bio-treatment
methodologies for archaeological monuments and sites.

    Abeer ElHagrassy is an associated professor in conservation of inorganic materi-
als and in applied microbiology in cultural heritage at Fayoum University Egypt. She is
a head manager of the Research and Conservation Center, at the Faculty of Archaeol-
ogy, Fayoum University. She is a scientific consultant for Takween and Megawra Com-
pany in Conservation Projects. She is a certified trainer at the Ministry of Antiquities,
Egypt. She is a reviewer for Egyptian Biophysic journals and the international journal
of microbiology. Her main research interests are in applied microbiology in environ-
ments, nanomaterial for the conservation of culture heritage, biodeterioration and bio-
degradation of monuments, bio treatment methods for archaeological monuments.


   Summary

    One bacterial species and five fungi were isolated from the mural paintings of the
Egyptian tomb of Khety (Tomb No. 17, 11th Dyn.). The antimicrobial activity of lemon
and thyme essential oils (EOs) diluted in ethanol 70% was evaluated against the bac-
terial isolate (related to the genus Alcaligenes and the species faecalis identified using
16 rRNA gene sequencing) and the most predominant fungi in the tomb, Aspergillus ni-


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                                                                                                                                                       ger, Penicillium cyclopium and Fusarium Solani on agar plates using the disc diffusion
M.D. Albasil, G. Mahgoub, A. ElHagrassy, A.M. Reyad - Evaluating the antimicrobial activity of essential oils in the conservation of mural paintings



                                                                                                                                                       method. Fragments were taken from the mural painting layers for analysis and exam-
                                                                                                                                                       ination to prepare replicas (5×5cm) similar to the archaeological mural painting and
                                                                                                                                                       were artificially aged. Antimicrobial activity of the two studied EOs was evaluated
                                                                                                                                                       against the bacterial isolate and the three studied fungi on replicas. Interestingly, our
                                                                                                                                                       results revealed that the thyme oil at a concentration of 15% is generally more efficient
                                                                                                                                                       than lemon EO against the three studied fungi and, in contrast, the lemon oil was more
                                                                                                                                                       efficient against the bacterium than fungi. Moreover, colorimetric measurements
                                                                                                                                                       proved that no notable changes occurred to the replica after treatment with the two
                                                                                                                                                       tested EOs in 10% and 15%. From the GC-MS analysis, the volatile profile of the lem-
                                                                                                                                                       on oil revealed that it is mainly composed of monoterpenes, with citral, 6-exohydroxy
                                                                                                                                                       camphene, and Fenchone as the major compounds. Our results showed that thymol
                                                                                                                                                       (44.38%) is the major compound for the thyme oil.


                                                                                                                                                          Riassunto

                                                                                                                                                           Dai dipinti murali della tomba egizia di Khety (tomba n. 17, 11 din.) sono stati isola-
                                                                                                                                                       ti una specie batterica e cinque funghi. L’attività antimicrobica degli oli essenziali di li-
                                                                                                                                                       mone e timo (EO) diluiti in etanolo al 70% è stata valutata rispetto all’isolato batterico
                                                                                                                                                       (relativo al genere Alcaligenes e alla specie faecalis identificato utilizzando il sequen-
                                                                                                                                                       ziamento del gene 16 rRNA) e al fungo predominante nella tomba, Aspergillus niger,
                                                                                                                                                       Penicillium cyclopium e Fusarium Solani su piastre di agar, utilizzando il metodo della
                                                                                                                                                       diffusione del disco. Frammenti sono stati prelevati dagli strati della pittura murale per
                                                                                                                                                       effettuare analisi su repliche (5×5 cm), simili alla pittura murale archeologica, invec-
                                                                                                                                                       chiati artificialmente. L’attività antimicrobica dei due EO studiati è stata valutata rispet-
                                                                                                                                                       to all’isolato batterico e ai tre funghi studiati sulle repliche. È interessante notare che ri-
                                                                                                                                                       sultati hanno rivelato che l’olio di timo, a una concentrazione del 15%, è generalmente
                                                                                                                                                       più efficiente del limone contro i tre funghi studiati e, al contrario, l’olio di limone è più
                                                                                                                                                       efficace contro il batterio rispetto ai funghi. Inoltre le indagini colorimetriche non hanno
                                                                                                                                                       mostrato cambiamenti significativi nelle repliche dopo il trattamento con i due EO te-
                                                                                                                                                       stati con concentrazione del 10% e del 15%. L’analisi GC-MS ha rivelato che il profilo
                                                                                                                                                       volatile dell’olio di limone è composto principalmente da monoterpeni, con citrale,
                                                                                                                                                       6-esoidrossi canfene e Fenchone come composti principali. I risultati hanno mostrato
                                                                                                                                                       che il timolo (44,38%) è il composto principale per l’olio di timo.




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