- Email: [email protected]
Biogenic deterioration of Egyptian limestone monuments: treatment and conservation
Journal of Cultural Heritage 38 (2019) 118–125
Available online at
ScienceDirect www.sciencedirect.com
Original article
Biogenic deterioration of Egyptian limestone monuments: treatment and conservation Khaled Z. ElBaghdady a,∗ , Sahar T. Tolba a , Soha S. Houssien b a b
Microbiology Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt Microbiology Unit, Research and Conservation Center of antiquities, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 8 December 2018 Accepted 8 February 2019 Available online 2 March 2019 Keywords: Monuments Limestone Bacteria Deterioration Simulation Antibacterials
a b s t r a c t The aim of this research was to study the biodeterioration activity of bacteria on ancient Egyptian limestone monuments and their treatments. Specimens and swabs were collected from seven different archeological sites along Egypt. According to the results of bacterial count, high bacterial load was detected at Senusret I obelisk and Mosque of Elkadi Abd El Basset. Qualitative and quantitative analysis of calcium carbonate degradation showed that the most damaging isolates were Bacillus safensis 9K (MH370265) and Streptomyces rochei 50 (MH370266) with 20.9 and 25.6% of total amounts of CaCO3 degradation, respectively. Environmental scanning electron microscope/Energy dispersive X-ray spectroscopy (ESEM/EDX) and physical characters of stone simulation model revealed that these isolates caused morphological, physical and chemical changes. Among the natural and chemical antimicrobial agents applied directly on the infected models as simulators of treatment and conservation processes, cetrimonium (1 mg/mL), tetra ethyl ammonium bromide (0.6 mg/mL), cinnamon (1 mg/mL) and cinnamon (5 mg/mL) were found to be effective against the two bacterial isolates. Fourier Transform Infrared Spectroscopy (FTIR) analysis for artificially deteriorated stones revealed that the compounds were safe for direct application on limestone monuments when no visual or chemical changes in the stone structure were observed. © 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction Cultural Heritage plays a very important role in our life since it connects people to their certain social values, beliefs, religions and traditions. Ancient Egypt was regarded as the ‘‘state out of stone” because stone was the most important raw material used during the different periods of Pharaonic Egypt as well as GrecoRoman and Arab times [1]. Stone buildings located in tropical and sub-tropical regions throughout the world are particularly vulnerable to microbial deterioration due to the prevailing environmental conditions of temperature and humidity, which are suitable for microbial growth [2]. Microorganisms are able to obtain different elements for their metabolism, e.g., calcium, aluminum, silicon, iron and potassium, by biosolubilization of materials. Such microbial biosolubilization involves the production of organic and inorganic acids by the metabolic activities of algae, lichens, fungi and bacteria [3].This acidic deterioration is one of the common known biogeochemical mechanisms of rock decay. Researches demon-
∗ Corresponding author. E-mail address: [email protected] (K.Z. ElBaghdady). https://doi.org/10.1016/j.culher.2019.02.005 1296-2074/© 2019 Elsevier Masson SAS. All rights reserved.
strated that numerous heterotrophic microorganisms present on the stonework were able to utilize, for their growth, the airborne organic compounds settled on the stone surface which mainly arose from the incomplete combustion of fossil fuels [4]. This evidence indicated that heterotrophic microorganisms can act as first colonizers in the areas with a high level of organic pollutants [5]. Chemotrophic prokaryotes could contaminate stones down to 5 cm deep or even deeper [6]. Chemoheterotrophic bacteria from Phylum Firmicutes are frequently identified on surface as well as inside the stone artifacts. Strains of Bacillus spp. with their spore forming strategy of survival and broad range of utilized nutrients were some of the most found bacteria [7]. Several strains related to moderately halophilic Gram-positive bacteria were isolated from wall paintings surfaces and building materials [8]. Halotolerant bacteria (Kocuria spp., Micrococcus spp., Arthrobacter spp., Bacillus spp., Staphylococcus spp., Paenibacillus spp.) were remarkable stone degrading isolates [9]. Chemolithoautotrophic bacteria present as epi-, hypo- or endolithic microbiota use CO2 or HCO3 - as carbon source and inorganic compounds as electron donors. Inorganic sources of energy mainly consist of sulfur compounds (S2 O3-2 , H2 S) and nitrogen compounds (NO2 -, NH3 ). As a result of their activity, inorganic acids (HNO3 , HNO2 , H2 SO4 ) were produced with dissolv-
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
ing effects on hosting material [6,9]. Populations of sulfur-oxidizing (Thiobacillus spp.) and nitrogen-cycling bacteria (Nitrobacter spp., Nitrosomonas spp., Nitrospira spp.) had been detected in samples of stones originated from various monuments [9]. Gram-positive actinobacteria are well represented in endolithic communities around the world [10]. Several strains of Streptomyces species have been isolated from ancient Egyptian mural paintings and stone support [11]. Streptomyces species were pointed as one of the main agents causing color change of tomb paintings by producing a range of acids (oxalic, citric, and succinic acid), biopigments (melanin) and hydrogen sulfide. Accordingly, the approach to control biodeterioration must be a polyphasic and interdisciplinary that considers the history and condition of the artifact as well as physical and chemical damaging factors. Actions against microbial growth can be divided into two major categories: indirect control by altering environmental condition and direct control by mechanical removal of biodeteriogens, chemicals (biocides), natural (essential oils) and physical eradicative methods. This study aimed to detect and identify potent bacteria responsible for biodeterioration of some ancient Egyptian limestone monuments, and then to treating the causative bacteria with safe antimicrobial agents for conserving the monuments. 2. Materials and methods 2.1. Sampling sites Deteriorated limestone materials were collected from seven historic Egyptian sites including; Seti I tomb at Luxor, Senusret I obelisk of Al Mattaryia district, Giza pyramid complex and related tombs, store of National Museum of Egyptian Civilization (NMEC), Mosque of Elkadi Abd El Basset (Gamaliya), Roman Amphitheatre of Alexandria and Ismailia Museum of Antiquities Fig. 1A-C. 2.2. Sampling and isolation of bacteria and actinobacteria Stone samples and cotton swabs were collected from different spots that showed aspects of stone decay at the study sites. The swabs were collected from deteriorated sites of the archeological objects then directly subcultured onto nutrient and inorganic salt starch agar [12]. The decayed stones were scratched and dumped into sterile Eppendorf tubes, serial dilutions were made and plated onto each of the two media [13]. Plates were incubated at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. The colonies were purified for further analyses. 2.3. Ability of isolates to degrade calcium carbonate The ability to produce acids as indicator for stone colonization and degradation was detected by culturing the pure isolates on plates containing mineral agar medium of pH 8 supplemented with 2% (w/v) calcium carbonate [14]. The plates were incubated for 2 days at 37 ◦ C for bacteria and 7 days at 28 ◦ C for actinobacteria. Isolates that were able to grow were selected for further study. 2.4. Quantitative assay for calcium carbonate degradation Mineral broth medium supplemented with CaCO3 (2% w/v) was inoculated with 106 CFU/mL of the selected isolates and incubated for 7 and 30 days at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively, in a shaking incubator (170 rpm) [15]. After incubation, the cultures were filtered through Whatman filter paper No. 1. The CaCO3 residue that trapped on filter paper was dissolved into 30 mL/1M HCl to dissolve the non-utilized CaCO3 , leaving excess HCl that expresses the microbial degradation of CaCO3 . The excess
119
HCl was titrated against 1M NaOH using phenolphthalein as indicator. Neutralization point attained when the solution turned from colorless to faint pink indicating that all excess HCl reacted with NaOH. Standard curve of CaCO3 was plotted using different calcium carbonate weights (0.2–1 g/30 mL/1M HCl) for calculation of calcium degradation percentages by the selected isolates [16].
2.5. Acid and melanin production Acid and melanin production were the most common mechanical and esthetical problems of stone monuments. The ability of isolates to produce acid was detected by changing the glucose bromocresol agar color from purple to yellow, while melanin production was detected by production of dark pigment after growing on tyrosine agar [17].
2.6. Conventional and molecular identification of isolates Morphological and cultural characteristics of actinobacteria isolates were studied by surface inoculation of the selected isolates onto inorganic salt starch agar, glycerol asparagine agar, yeast-malt extract agar and soybean meal agar [17]. Microscopic examination of actinobacteria was carried out using slide culture technique [18]. Bacterial isolates were subcultured on nutrient agar medium and Gram stained. PCR technique was carried out to partially amplify 16S rRNA gene for the selected isolates. A loopful of overnight grown cells was transferred to 50 L TE buffer and boiled for 5 min. Then, 1 L of cell suspension was used as DNA template for PCR reaction. PCR was performed using Fermentus Dream Taq Mastermix according to the instruction manual. The universal primers PA (5’-AGAGTTTGATCCTGGCTCAG-3’) and 517R (5’-ATTACCGCGGCTGCTGG-3’) were used to amplify 500 bp [19]. The reaction mixture for PCR was Mastermix 12.5 L, PA (10 M) 0.5 L, 517R (10 M) 0.5 L, DNA template 1 L and distilled water 10.5 L. PCR was performed using thermal cycler (Applied Biosystem 2720). The PCR conditions were adjusted to 5 min. for initial denaturation at 94 ◦ C and then 35 cycles of 1 min at 94 ◦ C, 1 min. at 54 ◦ C, and 1 min. at 72 ◦ C, and finally 10 min. at 72 ◦ C. The PCR products were subjected to electrophorasis on 1% agarose gel in TAE buffer and visualized under UV light (312 nm). Images were picked using GelDoc. Ingenius 3. PCR products were purified by Qiagen extraction kit according to the manufacturer instructions before applying to DNA sequencer. Sequencing was performed by GATC Company, Sigma, Cairo, Egypt. Nucleotide sequences were analyzed using Blastn [20] and submitted to GenBank to obtain the accession numbers.
2.7. Minimum inhibitory concentration (MIC) of natural and synthetic compounds Antimicrobial activities of both natural (cinnamon, clove, pepper mint, lavender, camphor and thyme essential oil) and synthetic compounds (para chloro meta cresol (PCMC), tetra ethyl ammonium bromide (TEAB) and cetrimonium) were applied against the selected potent isolates. Different concentrations (1- 0.03% in 95% ethyl alcohol) of active compound were prepared for minimum inhibitory concentration assessment. Well agar diffusion assay was carried out on nutrient agar and inorganic salt starch agar for isolates. Each plate was inoculated with 105 CFU/ml and the MIC was determined as the lowest concentration that inhibited growth of the isolates [21,22].
120
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
Fig. 1. Deteriorated surfaces of different limestone monuments. A. and C. From National Museum of Egyptian Civilization (NMEC) store, Cairo, Egypt. B. Senusret I obelisk at Al-Matariyah district. D. Museum of Ismailia Antiquities.
2.8. Testing effect of active compounds on limestone by ATR-FTIR Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis was used to assess the effect of tested antimicrobial agents on stone components. Natural limestone cubes (previously sterilized by autoclaving) were immersed in MIC of natural and synthetic antimicrobial compounds for 24 h. then, spectral analysis (600 to 4000 cm-1 ) was recorded using Vertex 70 Spectrometer [23].
• porosity = Density × Water absorption; • compressive strength = (Mechanical width).
load
×102)/(length ×
2.9.2. Environmental scanning electron microscope (ESEM/EDX) of simulated limestone cubes Simulated stones were subjected to ESEM/EDX (JEOL- JSM 5500 L V, Japan) to determine morphological and mineral changes after artificial deterioration of limestones [25].
2.9. Simulation of deterioration process Cubes (5 cm3 ) of natural limestone were used as short term simulation model. The cubes were autoclaved at 121 ◦ C for 20 min. then artificially inoculated on their surfaces with 50 mL of freshly prepared spore suspension (1 × 105 CFU/mL) of the isolates in sterile beaker under aseptic conditions, then sealed by parafilm tape and incubated in a shaking incubator (120 rpm) for one month at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. On the other hand, the inoculated cubes were used as control samples [15]. The infected cubes were visually examined every week for signs of deterioration. After incubation period, the two following tests were performed to detect the biodeterioration signs. 2.9.1. Measurements of physical characters of simulated limestone cubes Porosity, mechanical strength and water absorption were measured for the artificially deteriorated cubes using the following equations [24]: • water absorption = [(wet weight-dry weight)/ dry weight] ×100; • density = the weight/(length × width × height);
2.10. Simulation of treatment and conservation processes For simulation of treatment experiment, 6 sets of limestone cubes (each contained 3 cubes of 1 cm3 ) were used. Three sets were inoculated with bacterial spore suspension (1 × 104 CFU/mL) and the other 3 sets were inoculated with the actinobacterial spore suspension (1 × 104 CFU/mL). All sets were incubated for one month at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. After incubation periods, 4 sets were treated with MIC of the most active synthetic and natural compounds then incubated for 24 h at room temperature. The other 2 sets were used as controls (infected without treatment). For simulation of conservation, 4 sets were treated with the same active antimicrobial compounds (that was used for treatment model) and incubated for 24 h, after that, they were inoculated with the same potent isolates before incubation for a month at proper temperature. After incubation periods, each set was immersed into a saline solution, vortexed and subcultured on proper agar plates. The cultured plates were incubated for 24 h and 7 days at 37 and 28 ◦ C for bacteria and actinobacteria, respectively, then colony count was recorded for each set [24].
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
121
Fig. 2. Minimum inhibitory concentrations of active antimicrobials compounds against potent isolates of limestone monuments. PCMC: para chloro meta cresol and TEAB: tetra ethyl ammonium bromide. Bars represented standard error (n = 3).
2.11. Statistical analysis
3.5. Molecular identification of potent isolates
All statistical analysis in this study was carried out using Microsoft Excel 2016, Analysis Toolpack (Microsoft Corporation). All data were calculated from at least 3 replicates and the standard error for each datum was plotted on the graph.
The most potent actinobacterial isolate which identified by partial 16S rRNA gene sequencing was Streptomyces rochei 45AKS and submitted to GenBank under accession number MH370266, while the bacterial isolate was No 19 and identified as Bacillus safensis 9AKS (MH370265).
3. Experimental results
3.6. Minimum inhibitory concentration (MIC) of antimicrobial compounds
3.1. Microbial load of stone monuments Results recovered from solid decayed scratches of each site showed that Giza pyramid complex and Seti I tomb had the highest actinobacterial counts (13 × 104 and 8 × 104 ), while the lowest counts were recorded at Mosque of Judge Abd El Basset and National Museum of Egyptian Civilization (10 × 103 and 11 × 103 , respectively). On the other hand, Mosque of Judge Abd El Basset and Seti I tomb had the highest bacterial counts (85 × 104 and 47 × 104 , respectively), The lowest bacterial counts were recorded at Giza pyramid complex and related tomb, followed by Museum of Ismailia (11 × 103 and 10 × 104 , respectively). Results also showed that the mean counts of cotton swabs were in accordance with those obtained by serial dilutions.
3.2. Screening isolates for calcium carbonate degradation Highest percentage (75%) of bacterial isolates that was able to grow on calcium carbonate mineral medium was detected from Giza pyramid complex, followed by 66.6% from Roman amphitheater and 55% from Seti tomb. The lowest percentages were obtained from Mosque of Judge Abd El Basset (30.3%) and Senusret I obelisk (35.7%). All actinobacterial isolates were able to grow on calcium carbonate mineral medium. A total isolates of 43 actinobacteria and 32 bacteria were recovered from all sites.
3.3. Degradation of calcium carbonate isolates The highest percentages of calcium carbonate degradation by actinobacterial and bacterial isolates were 28.85% and 20.9%, respectively.
3.4. Detection of acid and melanin production All bacterial and actinobacterial isolates were found to be acid producers. While no bacterial isolate was able to produce melanin pigment, 33.3% of the actinobacteria isolates were able to do so.
Screening for antimicrobial activity of natural and synthetic compounds revealed that cinnamon, clove, thyme oil and all tested chemical compounds were active against both two potent isolates. The MICs of PCMC, TEAB and cetrimonium against Bacillus safensis 9AKS were 1, 2.5 and 1 mg/mL, while they were 1, 2.5 and 2.5 mg/mL for cinnamon, clove and thyme, respectively. PCMC, On the other hand, MICs of TEAB and cetrimonium against Streptomyces rochei 45AKS were 1, 0.6 and 0.6 mg/mL, while it was 5 mg/ml for each of the natural compounds (Fig. 2). 3.7. Effects of antimicrobial compounds on limestone As shown in (Fig. 3), ATR-FTIR analysis revealed that no chemical or visual changes were occurred on the limestone after treatment with any of the active antimicrobial compounds used. 3.8. Simulation of deterioration process Stones inoculated with either Bacillus safensis or Streptomyces rochei showed different visually deterioration signs including turbidity, powdering stones dissolution and dark pigmentation (Fig. 4). 3.8.1. Physical changes on simulated limestone cubes Various physical changes were observed on the stones infected by Streptomyces rochei and Bacillus safensis. Stone strength was reduced by 41.9% and 23.3% for Streptomyces sp. and Bacillus safensis, respectively. In addition, the porosity was reduced by 49.4% for Streptomyces rochei and 6.1% for Bacillus safensis (Fig.5). 3.8.2. Environmental scanning electron microscope (ESEM/EDX) of simulated limestone cubes ESEM revealed several deterioration features on the artificial inoculated limestone (Fig. 6). Disintegration of calcite crystal, as well as loss of binding materials between grains due to the microbial growth was observed. In addition, small fissures and cavities were formed within stone granules. EDX microanalysis of stone samples revealed that, the control consisted of calcium (Ca), silicon (Si), sodium (Na), aluminum (Al),
122
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
Fig. 3. FTIR analysis of control and treated limestone cubes. A. Control and treated with clove, cinnamon and thyme. B. Control and treated with cetrimonium, PCMC: para chloro meta cresol and TEAB: tetra ethyl ammonium bromide.
Fig. 4. Biodeterioration signs of limestone cubes. A. Inoculated limestone cube (control). B. Turbidity due to microbial activity. C. and D. pigmentation due to Bacillus safensis and Streptomyces rochei growth, respectively.
carbon (C) and (O) oxygen, meanwhile, analysis of infected samples by both strains demonstrated reduction of calcium weight which is the main constituent of limestone. On contrary, sodium and chlorine increased and also new elements appeared like iron and sulfur element suggesting new salt formation. 3.9. Simulation of treatment and conservation processes
Fig. 5. Reduction percentages in the stones physical characteristics caused by Bacillus safensis and Streptomyces rochei growth. Bars represented standard error (n = 3).
Bacillus safensis and Streptomyces rochei were inhibited by cetrimonium (1 mg/mL) and TEAB (0.6 mg/mL), while same amount of cinnamon oil inhibited 99.7% and 99.8% of the two isolates, respectively, when used as treatment after infection. A similar pattern was observed for conservation assessment as, cetrimonium (1 mg/mL) and TEAB (0.6 mg/mL) inhibited 99.6% and 98.9% of Bacillus safensis and Streptomyces rochei, respectively. Cinnamon oil (1 and 5 mg/mL) inhibited 99.1 and 93.1 of the isolates, respectively (Fig. 7).
Fig. 6. ESEM imaging of artificial inoculated limestone. A. Non-colonized stone (control). B. Stone inoculated with Bacillus safensis. C. Stone inoculated with Streptomyces rochei.
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
123
Fig. 7. Simulation of treatment and conserved processes of limestones inoculated with Bacillus safensis and Streptomyces rochei. (1) Cetrimonium against B. safensis inoculated stone; (2) Cinnamon oil against B. safensis inoculated stone; (3) TEAB (tetra ethyl ammonium bromide) against S. rochei inoculated stone and (4) Cinnamon against S. rochei inoculated stone. Bars represented standard error (n = 3).
4. Discussion Limestone has been used as a building material for many centuries in Egypt, representing the cultural heritage of mankind of each period. This study sheds light on the bacterial degradation at 7 different Egyptian archeological sites (upper and lower Egypt), as well as on the methods of treatment and conservation. Lower and Upper Egypt temperature and relative humidity are varied throughout the year consequently, produced different types of microbial infection [26]. Microbial counts of the studied sites revealed that bacteria were predominant over actinobacteria all except at Giza pyramid complex site, where actinobacteria was predominant. This was agreed with Eric et al [27] and Grigorevski-Limaa et al [28] who reported one fold increase in bacteria compared with actinobacteria in different touristic environments. Higher count of actinobacteria at Giza pyramid region was attributed to formation of spores that resist desiccation and starvation for long periods [29]. Previous results found that actinobacterial isolates were able to grow on calcium carbonate mineral medium with slightly alkaline medium (pH 7.8-8), which resemble the natural limestone [30]. The high salt tolerance of actinobacteria qualified them to persist and attack calcium carbonate consequently damaging stones [31]. High percentages of bacterial isolates that were able to grow on mineral medium (75, 66.6% and 55% at Giza pyramid complex, Roman amphitheater and Seti tomb, respectively) were due to low organic contaminants [32]. Other sites including Mosque of Judge Abd El Basset and Senusret I obelisk demonstrated low percentage of stone colonizing bacterial isolates. Mosque of Judge Abd El Basset, on the other hand, was a residential area which is highly polluted and had great sewage drainage problem as well as Senusret I obelisk which is polluted with garbage and fossil fuel [33]. Previous reports demonstrated that, numerous heterotrophic microorganisms present on the stonework can utilize, for their growth, the airborne organic pollutant settled on the stone surface and incomplete combustion of fossil fuel [5]. Acid and pigments production were very common mechanisms in the destruction of arts, where color changes to rock monuments are a real threat to cultural heritage, which is irreversible process [34]. In this study, both bacteria and actinobacteria were found to be acid producers, causing pitting, dissolution and powdering of
stone. Some actinobacterial isolates obtained from the sites under study were able to produce pigments, where no bacterial isolate was a melanin producer [35]. Most of bacterial isolates colonizing deteriorated stone monuments belonged to Gram positive bacteria represented by Bacillus spp. and Streptomyces spp, while Gram negative bacteria had low occurrence. This was in agreement with the previous study of AbdElkareem and Mohamed [36]. In addition, it was reported that the isolated carbonate-dissolving bacteria of limestone were affiliated to families Bacillaceae, Staphylococcaceae as well as Streptomyces species [37]. The most degrading bacterial isolate identified in this study, Bacillus safensis, was also previously recorded as contaminant on fresco which is a technique of mural painting (any piece of artwork painted or applied directly on a wall, ceiling or other surface) [38]. B. safensis is a Gram-positive, aerobic, mesophilic, chemoheterotrophic bacterium [39]. Satomi [40] and Kothari [41] have also reported the isolation of B. safensis strains from environments characterized with conditions like low moisture, high soil salinity, nutrient deficiency, high ultraviolet radiation and temperature, and strong winds, which are similar conditions to Seti tomb at Luxor and Giza pyramid complex site. Streptomyces rochei was considered as mesophilic, alkaliphilic, moderate salt tolerant in nature and strong acid producer from different carbon sources; all such characters enable it to grow within limestone monuments [42]. The present study was extended to determine the efficiency of some chemical and natural products against stone colonizing bacteria. Chemical (PCMC, TEAB and cetrimonium) and natural (cinnamon, clove, pepper mint, lavender, camphor and thyme) compounds were tested for antimicrobial activity. TEAB and cetrimonium were considered as the most common quaternary ammonium compounds in this regard, where they affect the cytoplasmic membrane and cause denaturation of proteins, resulting in leakage of intracellular components and death of bacteria [43]. PCMC was classified as a halo phenolic compound that has both halogen and hydroxyl groups that generally considered cellular poisons, reacting with a cell membrane and variety of macromolecules and microbial structures and inhibits fatty acid synthesis via their reactive hydroxyl and halogen groups [44]. Synthetic antimicrobial agents in this study such as PCMC, TEAB and cetrimonium inhibited the microbial growth at very low con-
124
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
centrations reached 0.03% (0.3 mg/mL), which was supported by earlier observations [45,46]. Essential oils including thymol and its isomer carvacrol, cinnamon and clove have been used throughout history as preservatives. Oregano and thyme essential oils have been shown to be strongly microbicidal against Gram positive and Gram negative bacteria as well as actinobacteria [47] via destabilizing the cytoplasmic membrane and acting as a proton exchanger [48]. Some studies [49,50] suggested that compounds penetrate the cell, where they interfere with cellular metabolism and phenols such as carvacrol and eugenol (main component of essential oil), disturb the cellular membrane and react with active sites of essential enzymes. Both thyme and cinnamon released high levels of carvacrol, thymol and cinnamaldehyde, respectively, during the first 3–6 h of incubation leading to bacterial inhibition [51]. Concerning natural antimicrobial compounds, cinnamon, clove and thyme have antibacterial activity at very low concentration (0.5% or 5 mg/mL) against almost rock colonizing bacteria, a study which agreed with that of Mironescu et al. [52]. FTIR confirmed safety of the tested antimicrobial compounds to stone material, since no variation or changes in stone chemical composition were observed on the simulated treated stone cubes with the effective MICs of both natural and chemical compounds. Consequently, they could be directly applied at any archeological sites as common antimicrobial compounds. Visual observation of deterioration sings at simulation experiment was agreed with Styriakova et al. [53] who recorded that organic acids production, bacterial colonization and the destruction of the mineral surfaces of grains greatly accelerated the release of mineral elements from rocks to solution, which appeared as turbidity and stone bleaching, although these processes take many years in nature. Dark pigmentation and stone discoloration was clearly obvious on stone surface, these observations were recorded by actinobacteria which tolerated high salt environment [54]. Melanin negative Streptomyces spp. produced different types of diffusible pigment (yellow to dark brown) affecting stone appearance [55]. The deterioration is a complex process that changes the mechanical properties and mineralogical composition of limestone. Concurrently, when gypsum or crust was formed in the pores of limestone it can serve as pore blocking cement [56], and thus it helps in the formation of a non-porous surface crust on the limestone, decreasing the porosity of stone and causes weakening of the host rock [57]. This explained the compressive strength and porosity calculations recorded in this study. Application of synthetic antimicrobial compounds show more bactericidal activities than that of natural one, which may due to high stability and penetration within stone surface especially when they used at control or conservation experiment. This also may be attributed to the high volatility of different essential oils causing smaller residual effect of them inside the stone which was affected by different conditions like oil concentration, texture and porosity of the stone, crust formation, aeration and temperature [58].
5. Conclusion In this study, the microbial load of deteriorated limestone monuments from different Egyptian archeological sites was recorded. The most potential biodeteriogens were identified as Bacillus safensis and Streptomyces rochei. Natural; cinnamon, clove, thyme and synthetic; PCMC, TEAB, certrimonium antimicrobial agents were able to treat and protect stones from the potent stone degrading isolates which could be applicable on archeological sites. Essential oils are effective, safe, available, and cheap which have no residual effect on stone which qualify them to be applied on archeological limestone objects.
Funding source This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1] D.D. Klemm, R. Klemm, The building stones of ancient Egypt - a gift of its geology, J. Afr. Earth Sci. 33 (2001) 63–642. [2] M.B.O. Ortega, C.C. Gaylarde, G.E. Englert, P.M. Gaylarde, Analysis of saltcontaining biofilms on limestone buildings of the Mayan culture at Edzna, Mexico, Geomicrobiol. J. 22 (2005) 261–268. [3] L.K. Herrera, C. Arroyave, P. Guiamet, S.G. Saravia, H. Videla, Biodeterioration of peridotite and other constructional materials in a building of the Colombian cultural heritage, Int. Biodeter. Biodegrad. 54 (2004) 135–141. [4] J.C. Saiz, The role of microorganisms in the removal of pollutants deposited onto historic buildings, Int. Biodeterior. Biodegrad. 40 (1997) 225–232. [5] E. Zanardini, P. Abbruscato, N. Ghedini, M. Realini, C. Sorlini, Influence of atmospheric pollutants on biodeterioration of stone, Int. Biodeterior. Biodegrad. 45 (2000) 35–42. [6] T. Warscheid, J. Braams, Biodeterioration of stone: a review, Int. Biodeterior. Biodegrad. 46 (2000) 343–368. [7] S. Scheerer, M.O. Ortega, C. Gaylarde, Microbial deterioration of stone monuments, Adv. Appl. Microbiol. 66 (2009) 97–139. ˜ [8] G. Pinar, C. Ramos, S. Rölleke, G.C. Schabereiter, D. Vybiral, W. Lubitz, E.B. Denner, Detection of indigenous Halobacillus populations in damaged ancient wall paintings and building materials: molecular monitoring and cultivation, Appl. Environ. Microbiol. 67 (10) (2001) 4891–4895. [9] L. Laiz, D. Recio, B. Hermosin, C. Saiz-Jimenez, Microbial communities in salt efflorescences, in: O. Ciferrio, P. Tiene, G. Mastromei (Eds.), On microbes and art, Proceedings of an international conference on microbiology and conservation, Kluwer Academic/Plenum Publishers, NewYork, 2000, pp. 77–88. [10] J.J. Walker, N.R. Pace, Endolithic microbial ecosystems, Annu. Rev. Microbiol. 61 (2007) 331–347. [11] M.E. Abdel-Haliem, A.A. Sakr, M.F. Ali, M.F. Ghaly, C. Sohlenkamp, Characterization of Streptomyces isolates causing colour changes of mural paintings in ancient Egyptian tombs, Microbiol. Res. 168 (7) (2013) 428–437. [12] E. Küster, Outline of a comparative study of criteria used in characterization of the actinomycetes, Int. Bull. Bact. Nomen. Taxon. 9 (1959) 97–104. [13] Y. Nuhoglu, E. Oguz, H. Uslu, A. Ozbek, B. Ipekoglu, I. Ocak, I. Hasenekoglu, The accelerating effects of the microorganisms on biodeterioration of stone monuments under air pollution and continental-cold climatic conditions in Erzurum, Turkey, Sci. Total Environ. 364 (2006) 272–283. [14] S. Evan, W. Ryan, M. Hasen, A. Schneegurt, Isolation and characterization of halotolerant soil fungi from the great salt plains of Oklahoma, Mycologia 34 (4) (2013) 329–341. [15] W. Song, N. Ogawa, T. Hatta, C.T. Oguchi, Y. Matsukura, Effect of Bacillus subtilis on granite weathering: a laboratory experiment, Catena, Biogeomicrobiol. 70 (3) (2007) 275–281. [16] W.W. Scott, Standard methods of chemical analysis, Fifth edition. Edited by N. Howell Furman, Ph.D. 2 vols, The Technical Press, Ltd.; New York. Price 6d, (1969) pp. 2617. [17] R. Atlas, Alphabetical listing of media, Handbook of microbiological media, 3rd Ed., CRC Press, Inc., Boca Raton, 2004. [18] M. Kawato, R. Shinobu, On Streptomyces herbaricolour, nov sp. supplement: a simple technique for the microscopic observation. Memoirs of the Osaka University of Liberal Arts and Education, B. Nat. Sci 8 (1960) 114–119. [19] H. Heuer, M. Krsek, P. Baker, K. Smalla, E.M. Wellington, Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients, App. Environ. Microbiol. 63 (8) (1997) 3233–3241. [20] S.F. Altschul, T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (17) (1997) 3389–3402. [21] M. Zyani, D. Mortabit, S. El Abed, A. Remmal, S.I. Koraichi, Antifungal activity of five plant essential oils against wood decay fungi isolated from an old house at the Medina of Fez, Int. Res. J. Microbiol. 2 (3) (2011) 104–108. [22] P. Bhatnagar, S.K. Jain, Antimicrobial activity of plant extract against fungi associated with monument deterioration of Gwalior Fort in India, European Academic Res. 2 (5) (2014) 6199–6210. [23] M.R. Derrick, C.S. Dusan, J.M. Landry, Infrared spectroscopy in conservation science, the Getty Conservation Institute, Los Angeles (1999). [24] J. Slusarek, The correlation of structure porosity and compressive strength of hardening cement material, Archit. Civil Eng. Environ. 3 (2010) 85–92. [25] G.D. Danilatos, V.N.E. Robinson, Principles of scanning electron microscopy at high specimen pressures, Scanning 2 (1979) 72–82. [26] M. Domroes, A. El-Tantawi, Recent temporal and spatial temperature changes in Egypt, Int. J. Climatol. 25 (2005) 51–63. [27] E. May, S. Papida, H. Abdulla, S. Taylerl, A. Dewedar, Comparative studies of communities on stone monuments in temperate and semi-arid climate, in: O. Ciferri, P. Tiano, G. Mastromei (Eds.), Of microbes and art: the role of microbial communities in the degradation and protection of cultural heritage, Kluwer Academic Publishers, New York, 2000, pp. 49–62.
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125 [28] A.L. Grigorevski-Limaa, R.G. Silva-Filhob, L.F. Linharesa, R.R.R. Coelhoa, Occurrence of actinomycetes in indoor air in Rio de Janeiro, Brazil. Build. Environ. 41 (11) (2006) 1540–1543. [29] E. May, Microbes on building stone: for good or ill? Culture 24 (2) (2003) 5–8. [30] R. Ghorbani, R. Greiner, H.A. Alikhani, J. Hamedi, B. Yakhchali, Distribution of actinomycetes in different soil ecosystems and effect of media composition on extracellular phosphatase activity, J. Soil Sci. Plant Nutr. 13 (1) (2013) 223–236. [31] H. Aria, On microorganisms in the tomb of Nefertari, Sci. Conser. 27 (1988) 19–20. [32] L.M. Suihko, L.H. Alakomi, A.A. Gorbushina, I. Fortune, M Saarela, Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments, Syst. Appl. Microbiol. 30 (2007) 494–508. [33] A.A. Ahmed, Land use change and deterioration of pharaonic of monuments in Upper Egypt, J. Eng. Sci. 37 (1) (2009) 161–177. [34] C. Giacobini, M.A. Decicco, I. Tiglie, G. Accardo, Actinomycetes and biodeterioration in the field of fine art, Biodeterioration 7 (1988) 418–427. [35] W.E. Krumbein, Colour changes of building stones and their direct and indirect biological causes, in: R. Delgado, F. Henriques, T. Jeremias (Eds.), Proceedings of the 7th International Congress on Deterioration and Conservation of Stone, Laboratorio Nacional de Engenharia Civil, Lisbon, Portugal, 1992, pp. 443–452. [36] E.A. Abd-Elkareem, R.M. Mohamed, Microbial deterioration of limestone of Sultan Hassan mosque, Cairo- Egypt and suggested treatment, Int. J. Chem. Tech. Res. 10 (5) (2017) 535–552. [37] G. Subrahmaniyam, R. Vaghela, N.P. Bhatt, G. Archana, Carbonate-dissolving bacteria from ‘Miliolite’, a bioclastic limestone, from Gopnath, Gujarat, Western India, Microbes Environ 27 (3) (2012) 334–337. [38] K.M. Reza, N. Ashrafalsadat, R.M. Reza, N. Taher, N. Ali, Isolation and molecular identification of extracellular lipase-producing Bacillus species from soil, Annals Biol, Res. 5 (2014) 132–139. [39] C.E. Raja, K. Omine, Arsenic, boron and salt resistant Bacillus safensis MS11 isolated from Mongolia desert soil, Afr. J. Biotechnol. 11 (2012) 2267–2275. [40] M. Satomi, T. Myron, L. Duc, K. Venkateswaran, Bacillus safensis sp. nov., isolated from spacecraft and assembly facility surfaces, Int. J. Sys, Evol. Microbiol. 56 (2006) 1735–1740. [41] V.V. Kothari, R.K. Kothari, C.R. Kothari, V.D. Bhatt, N.M. Nathani, P.G. Koringa, C.G. Joshi, B.R.M. Vyas, Genome sequence of salt-tolerant Bacillus safensis strain VK, isolated from saline desert area of Gujarat, India, Genome Announc. 1 (5) (2013) e00671–13. [42] R.N. Gopi, D.P.N. Ramakrishna, S.V. Rajagopal, Optimization of culture conditions of Streptomyces rochei (MTCC 10109) for the production of antimicrobial metabolites, Egypt. J. Biol. 13 (2011) 21–29.
125
[43] R. Xuehong, L. Jie, Smart anti-microbial composite coatings for textiles and plastics in: Smart composite coatings and membranes, Woodhead Publishing Ser. Composites Sci Eng (2016) 235–259. [44] G. McDonnell, Sterilization and Disinfection, Encyclopedia of Microbiology (Third Edition) (2009) 529–548. [45] S. Burt, Essential oils: their antibacterial properties and potential applications in foods a review, Int. J. Food Microbiol. 94 (3) (2004) 223–253. [46] N. Matan, H. Rimkeeree, A.J. Mawson, P. Chompreeda, V. Haruthaithanasan, M. Parker, Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions, Int. J. Food Microbiol. 107 (2) (2006) 180–185. [47] T. Nakatsu, A.L. Lupo, J.W. Chinn, R.K.L. Kang, Biological activity of essential oils and their constituents, Studies Nat. Prod. Chem 21 (2000) 571–631. [48] A. Ultee, E.P.W. Kets, M. Alberda, F.A. Hoekstra, E.J. Smid, Adaptation of the food-borne pathogen Bacillus cereus to carvacrol, Arch. Microbiol. 174 (2000) 233–238. [49] L.S. Ooi, S.L. Kam, H. Wang, E.Y. Wong, V.E. Ooi, Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume, Am. J. Chin. Med. 34 (3) (2006) 511–522. [50] P. López, C. Sánchez, R. Batlle, C. Nerín, Development of flexible antimicrobial films using essential oils as active agents, J. Agric. Food Chem. 55 (21) (2007) 8814–8824. [51] E. Pinto, L. Vale-Silva, C. Cavaleiro, L. Salgueiro, Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species, J. Med. Microbiol. 58 (11) (2009) 1454–1462. [52] M. Mironescu, C. Georgescu, L. Oprean, Comparative sporicidal effects of volatile oils, J. Agroalimentary Process. Technol. 15 (3) (2009) 361–365. [53] I. Styriakova, I. Styriak, H. Oberhansli, Rock weathering by indigenous heterotrophic bacteria of Bacillus spp. at different temperature: a laboratory experiment, Mineral. Petrol. 105 (2012) 135–144. [54] H. Abdulla, E. May, M. Bahgat, A. Dewedar, Characterisation of actinomycetes isolated from ancient stone and their potential for deterioration, Pol. J. Microbiol. 57 (3) (2008) 213–220. [55] M. Oskay, Antifungal and antibacterial compounds from Streptomyces strains, Afr. J. Biotechnol. 8 (13) (2009) 3007–3017. [56] Á. Torok, Surface strength and mineralogy of weathering crusts on limestone buildings in Budapest, Build. Environ. 38 (9) (2003) 1185–1192. [57] Á. Torok, R. Rozgonyi, Mineralogy and morphology of salt crusts on porous limestone in urban environment, Environ. Geol. 46 (3) (2004) 323–339. [58] M.E. Young, H.L. Alakomi, I. Fortune, A.A. Gorbushina, W.E.I Krumbein, C. McCullagh, P. Robertson, M. Saarela, J. Valero, M. Vendrell, Development of a biocidal treatment regime to inhibit biological growths on cultural heritage, Environ. Geol. 56 (2008) 631–641.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Biogenic deterioration of Egyptian limestone monuments: treatment and conservation Khaled Z. ElBaghdady a,∗ , Sahar T. Tolba a , Soha S. Houssien b a b
Microbiology Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt Microbiology Unit, Research and Conservation Center of antiquities, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 8 December 2018 Accepted 8 February 2019 Available online 2 March 2019 Keywords: Monuments Limestone Bacteria Deterioration Simulation Antibacterials
a b s t r a c t The aim of this research was to study the biodeterioration activity of bacteria on ancient Egyptian limestone monuments and their treatments. Specimens and swabs were collected from seven different archeological sites along Egypt. According to the results of bacterial count, high bacterial load was detected at Senusret I obelisk and Mosque of Elkadi Abd El Basset. Qualitative and quantitative analysis of calcium carbonate degradation showed that the most damaging isolates were Bacillus safensis 9K (MH370265) and Streptomyces rochei 50 (MH370266) with 20.9 and 25.6% of total amounts of CaCO3 degradation, respectively. Environmental scanning electron microscope/Energy dispersive X-ray spectroscopy (ESEM/EDX) and physical characters of stone simulation model revealed that these isolates caused morphological, physical and chemical changes. Among the natural and chemical antimicrobial agents applied directly on the infected models as simulators of treatment and conservation processes, cetrimonium (1 mg/mL), tetra ethyl ammonium bromide (0.6 mg/mL), cinnamon (1 mg/mL) and cinnamon (5 mg/mL) were found to be effective against the two bacterial isolates. Fourier Transform Infrared Spectroscopy (FTIR) analysis for artificially deteriorated stones revealed that the compounds were safe for direct application on limestone monuments when no visual or chemical changes in the stone structure were observed. © 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction Cultural Heritage plays a very important role in our life since it connects people to their certain social values, beliefs, religions and traditions. Ancient Egypt was regarded as the ‘‘state out of stone” because stone was the most important raw material used during the different periods of Pharaonic Egypt as well as GrecoRoman and Arab times [1]. Stone buildings located in tropical and sub-tropical regions throughout the world are particularly vulnerable to microbial deterioration due to the prevailing environmental conditions of temperature and humidity, which are suitable for microbial growth [2]. Microorganisms are able to obtain different elements for their metabolism, e.g., calcium, aluminum, silicon, iron and potassium, by biosolubilization of materials. Such microbial biosolubilization involves the production of organic and inorganic acids by the metabolic activities of algae, lichens, fungi and bacteria [3].This acidic deterioration is one of the common known biogeochemical mechanisms of rock decay. Researches demon-
∗ Corresponding author. E-mail address: [email protected] (K.Z. ElBaghdady). https://doi.org/10.1016/j.culher.2019.02.005 1296-2074/© 2019 Elsevier Masson SAS. All rights reserved.
strated that numerous heterotrophic microorganisms present on the stonework were able to utilize, for their growth, the airborne organic compounds settled on the stone surface which mainly arose from the incomplete combustion of fossil fuels [4]. This evidence indicated that heterotrophic microorganisms can act as first colonizers in the areas with a high level of organic pollutants [5]. Chemotrophic prokaryotes could contaminate stones down to 5 cm deep or even deeper [6]. Chemoheterotrophic bacteria from Phylum Firmicutes are frequently identified on surface as well as inside the stone artifacts. Strains of Bacillus spp. with their spore forming strategy of survival and broad range of utilized nutrients were some of the most found bacteria [7]. Several strains related to moderately halophilic Gram-positive bacteria were isolated from wall paintings surfaces and building materials [8]. Halotolerant bacteria (Kocuria spp., Micrococcus spp., Arthrobacter spp., Bacillus spp., Staphylococcus spp., Paenibacillus spp.) were remarkable stone degrading isolates [9]. Chemolithoautotrophic bacteria present as epi-, hypo- or endolithic microbiota use CO2 or HCO3 - as carbon source and inorganic compounds as electron donors. Inorganic sources of energy mainly consist of sulfur compounds (S2 O3-2 , H2 S) and nitrogen compounds (NO2 -, NH3 ). As a result of their activity, inorganic acids (HNO3 , HNO2 , H2 SO4 ) were produced with dissolv-
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
ing effects on hosting material [6,9]. Populations of sulfur-oxidizing (Thiobacillus spp.) and nitrogen-cycling bacteria (Nitrobacter spp., Nitrosomonas spp., Nitrospira spp.) had been detected in samples of stones originated from various monuments [9]. Gram-positive actinobacteria are well represented in endolithic communities around the world [10]. Several strains of Streptomyces species have been isolated from ancient Egyptian mural paintings and stone support [11]. Streptomyces species were pointed as one of the main agents causing color change of tomb paintings by producing a range of acids (oxalic, citric, and succinic acid), biopigments (melanin) and hydrogen sulfide. Accordingly, the approach to control biodeterioration must be a polyphasic and interdisciplinary that considers the history and condition of the artifact as well as physical and chemical damaging factors. Actions against microbial growth can be divided into two major categories: indirect control by altering environmental condition and direct control by mechanical removal of biodeteriogens, chemicals (biocides), natural (essential oils) and physical eradicative methods. This study aimed to detect and identify potent bacteria responsible for biodeterioration of some ancient Egyptian limestone monuments, and then to treating the causative bacteria with safe antimicrobial agents for conserving the monuments. 2. Materials and methods 2.1. Sampling sites Deteriorated limestone materials were collected from seven historic Egyptian sites including; Seti I tomb at Luxor, Senusret I obelisk of Al Mattaryia district, Giza pyramid complex and related tombs, store of National Museum of Egyptian Civilization (NMEC), Mosque of Elkadi Abd El Basset (Gamaliya), Roman Amphitheatre of Alexandria and Ismailia Museum of Antiquities Fig. 1A-C. 2.2. Sampling and isolation of bacteria and actinobacteria Stone samples and cotton swabs were collected from different spots that showed aspects of stone decay at the study sites. The swabs were collected from deteriorated sites of the archeological objects then directly subcultured onto nutrient and inorganic salt starch agar [12]. The decayed stones were scratched and dumped into sterile Eppendorf tubes, serial dilutions were made and plated onto each of the two media [13]. Plates were incubated at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. The colonies were purified for further analyses. 2.3. Ability of isolates to degrade calcium carbonate The ability to produce acids as indicator for stone colonization and degradation was detected by culturing the pure isolates on plates containing mineral agar medium of pH 8 supplemented with 2% (w/v) calcium carbonate [14]. The plates were incubated for 2 days at 37 ◦ C for bacteria and 7 days at 28 ◦ C for actinobacteria. Isolates that were able to grow were selected for further study. 2.4. Quantitative assay for calcium carbonate degradation Mineral broth medium supplemented with CaCO3 (2% w/v) was inoculated with 106 CFU/mL of the selected isolates and incubated for 7 and 30 days at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively, in a shaking incubator (170 rpm) [15]. After incubation, the cultures were filtered through Whatman filter paper No. 1. The CaCO3 residue that trapped on filter paper was dissolved into 30 mL/1M HCl to dissolve the non-utilized CaCO3 , leaving excess HCl that expresses the microbial degradation of CaCO3 . The excess
119
HCl was titrated against 1M NaOH using phenolphthalein as indicator. Neutralization point attained when the solution turned from colorless to faint pink indicating that all excess HCl reacted with NaOH. Standard curve of CaCO3 was plotted using different calcium carbonate weights (0.2–1 g/30 mL/1M HCl) for calculation of calcium degradation percentages by the selected isolates [16].
2.5. Acid and melanin production Acid and melanin production were the most common mechanical and esthetical problems of stone monuments. The ability of isolates to produce acid was detected by changing the glucose bromocresol agar color from purple to yellow, while melanin production was detected by production of dark pigment after growing on tyrosine agar [17].
2.6. Conventional and molecular identification of isolates Morphological and cultural characteristics of actinobacteria isolates were studied by surface inoculation of the selected isolates onto inorganic salt starch agar, glycerol asparagine agar, yeast-malt extract agar and soybean meal agar [17]. Microscopic examination of actinobacteria was carried out using slide culture technique [18]. Bacterial isolates were subcultured on nutrient agar medium and Gram stained. PCR technique was carried out to partially amplify 16S rRNA gene for the selected isolates. A loopful of overnight grown cells was transferred to 50 L TE buffer and boiled for 5 min. Then, 1 L of cell suspension was used as DNA template for PCR reaction. PCR was performed using Fermentus Dream Taq Mastermix according to the instruction manual. The universal primers PA (5’-AGAGTTTGATCCTGGCTCAG-3’) and 517R (5’-ATTACCGCGGCTGCTGG-3’) were used to amplify 500 bp [19]. The reaction mixture for PCR was Mastermix 12.5 L, PA (10 M) 0.5 L, 517R (10 M) 0.5 L, DNA template 1 L and distilled water 10.5 L. PCR was performed using thermal cycler (Applied Biosystem 2720). The PCR conditions were adjusted to 5 min. for initial denaturation at 94 ◦ C and then 35 cycles of 1 min at 94 ◦ C, 1 min. at 54 ◦ C, and 1 min. at 72 ◦ C, and finally 10 min. at 72 ◦ C. The PCR products were subjected to electrophorasis on 1% agarose gel in TAE buffer and visualized under UV light (312 nm). Images were picked using GelDoc. Ingenius 3. PCR products were purified by Qiagen extraction kit according to the manufacturer instructions before applying to DNA sequencer. Sequencing was performed by GATC Company, Sigma, Cairo, Egypt. Nucleotide sequences were analyzed using Blastn [20] and submitted to GenBank to obtain the accession numbers.
2.7. Minimum inhibitory concentration (MIC) of natural and synthetic compounds Antimicrobial activities of both natural (cinnamon, clove, pepper mint, lavender, camphor and thyme essential oil) and synthetic compounds (para chloro meta cresol (PCMC), tetra ethyl ammonium bromide (TEAB) and cetrimonium) were applied against the selected potent isolates. Different concentrations (1- 0.03% in 95% ethyl alcohol) of active compound were prepared for minimum inhibitory concentration assessment. Well agar diffusion assay was carried out on nutrient agar and inorganic salt starch agar for isolates. Each plate was inoculated with 105 CFU/ml and the MIC was determined as the lowest concentration that inhibited growth of the isolates [21,22].
120
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
Fig. 1. Deteriorated surfaces of different limestone monuments. A. and C. From National Museum of Egyptian Civilization (NMEC) store, Cairo, Egypt. B. Senusret I obelisk at Al-Matariyah district. D. Museum of Ismailia Antiquities.
2.8. Testing effect of active compounds on limestone by ATR-FTIR Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis was used to assess the effect of tested antimicrobial agents on stone components. Natural limestone cubes (previously sterilized by autoclaving) were immersed in MIC of natural and synthetic antimicrobial compounds for 24 h. then, spectral analysis (600 to 4000 cm-1 ) was recorded using Vertex 70 Spectrometer [23].
• porosity = Density × Water absorption; • compressive strength = (Mechanical width).
load
×102)/(length ×
2.9.2. Environmental scanning electron microscope (ESEM/EDX) of simulated limestone cubes Simulated stones were subjected to ESEM/EDX (JEOL- JSM 5500 L V, Japan) to determine morphological and mineral changes after artificial deterioration of limestones [25].
2.9. Simulation of deterioration process Cubes (5 cm3 ) of natural limestone were used as short term simulation model. The cubes were autoclaved at 121 ◦ C for 20 min. then artificially inoculated on their surfaces with 50 mL of freshly prepared spore suspension (1 × 105 CFU/mL) of the isolates in sterile beaker under aseptic conditions, then sealed by parafilm tape and incubated in a shaking incubator (120 rpm) for one month at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. On the other hand, the inoculated cubes were used as control samples [15]. The infected cubes were visually examined every week for signs of deterioration. After incubation period, the two following tests were performed to detect the biodeterioration signs. 2.9.1. Measurements of physical characters of simulated limestone cubes Porosity, mechanical strength and water absorption were measured for the artificially deteriorated cubes using the following equations [24]: • water absorption = [(wet weight-dry weight)/ dry weight] ×100; • density = the weight/(length × width × height);
2.10. Simulation of treatment and conservation processes For simulation of treatment experiment, 6 sets of limestone cubes (each contained 3 cubes of 1 cm3 ) were used. Three sets were inoculated with bacterial spore suspension (1 × 104 CFU/mL) and the other 3 sets were inoculated with the actinobacterial spore suspension (1 × 104 CFU/mL). All sets were incubated for one month at 37 ◦ C and 28 ◦ C for bacteria and actinobacteria, respectively. After incubation periods, 4 sets were treated with MIC of the most active synthetic and natural compounds then incubated for 24 h at room temperature. The other 2 sets were used as controls (infected without treatment). For simulation of conservation, 4 sets were treated with the same active antimicrobial compounds (that was used for treatment model) and incubated for 24 h, after that, they were inoculated with the same potent isolates before incubation for a month at proper temperature. After incubation periods, each set was immersed into a saline solution, vortexed and subcultured on proper agar plates. The cultured plates were incubated for 24 h and 7 days at 37 and 28 ◦ C for bacteria and actinobacteria, respectively, then colony count was recorded for each set [24].
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
121
Fig. 2. Minimum inhibitory concentrations of active antimicrobials compounds against potent isolates of limestone monuments. PCMC: para chloro meta cresol and TEAB: tetra ethyl ammonium bromide. Bars represented standard error (n = 3).
2.11. Statistical analysis
3.5. Molecular identification of potent isolates
All statistical analysis in this study was carried out using Microsoft Excel 2016, Analysis Toolpack (Microsoft Corporation). All data were calculated from at least 3 replicates and the standard error for each datum was plotted on the graph.
The most potent actinobacterial isolate which identified by partial 16S rRNA gene sequencing was Streptomyces rochei 45AKS and submitted to GenBank under accession number MH370266, while the bacterial isolate was No 19 and identified as Bacillus safensis 9AKS (MH370265).
3. Experimental results
3.6. Minimum inhibitory concentration (MIC) of antimicrobial compounds
3.1. Microbial load of stone monuments Results recovered from solid decayed scratches of each site showed that Giza pyramid complex and Seti I tomb had the highest actinobacterial counts (13 × 104 and 8 × 104 ), while the lowest counts were recorded at Mosque of Judge Abd El Basset and National Museum of Egyptian Civilization (10 × 103 and 11 × 103 , respectively). On the other hand, Mosque of Judge Abd El Basset and Seti I tomb had the highest bacterial counts (85 × 104 and 47 × 104 , respectively), The lowest bacterial counts were recorded at Giza pyramid complex and related tomb, followed by Museum of Ismailia (11 × 103 and 10 × 104 , respectively). Results also showed that the mean counts of cotton swabs were in accordance with those obtained by serial dilutions.
3.2. Screening isolates for calcium carbonate degradation Highest percentage (75%) of bacterial isolates that was able to grow on calcium carbonate mineral medium was detected from Giza pyramid complex, followed by 66.6% from Roman amphitheater and 55% from Seti tomb. The lowest percentages were obtained from Mosque of Judge Abd El Basset (30.3%) and Senusret I obelisk (35.7%). All actinobacterial isolates were able to grow on calcium carbonate mineral medium. A total isolates of 43 actinobacteria and 32 bacteria were recovered from all sites.
3.3. Degradation of calcium carbonate isolates The highest percentages of calcium carbonate degradation by actinobacterial and bacterial isolates were 28.85% and 20.9%, respectively.
3.4. Detection of acid and melanin production All bacterial and actinobacterial isolates were found to be acid producers. While no bacterial isolate was able to produce melanin pigment, 33.3% of the actinobacteria isolates were able to do so.
Screening for antimicrobial activity of natural and synthetic compounds revealed that cinnamon, clove, thyme oil and all tested chemical compounds were active against both two potent isolates. The MICs of PCMC, TEAB and cetrimonium against Bacillus safensis 9AKS were 1, 2.5 and 1 mg/mL, while they were 1, 2.5 and 2.5 mg/mL for cinnamon, clove and thyme, respectively. PCMC, On the other hand, MICs of TEAB and cetrimonium against Streptomyces rochei 45AKS were 1, 0.6 and 0.6 mg/mL, while it was 5 mg/ml for each of the natural compounds (Fig. 2). 3.7. Effects of antimicrobial compounds on limestone As shown in (Fig. 3), ATR-FTIR analysis revealed that no chemical or visual changes were occurred on the limestone after treatment with any of the active antimicrobial compounds used. 3.8. Simulation of deterioration process Stones inoculated with either Bacillus safensis or Streptomyces rochei showed different visually deterioration signs including turbidity, powdering stones dissolution and dark pigmentation (Fig. 4). 3.8.1. Physical changes on simulated limestone cubes Various physical changes were observed on the stones infected by Streptomyces rochei and Bacillus safensis. Stone strength was reduced by 41.9% and 23.3% for Streptomyces sp. and Bacillus safensis, respectively. In addition, the porosity was reduced by 49.4% for Streptomyces rochei and 6.1% for Bacillus safensis (Fig.5). 3.8.2. Environmental scanning electron microscope (ESEM/EDX) of simulated limestone cubes ESEM revealed several deterioration features on the artificial inoculated limestone (Fig. 6). Disintegration of calcite crystal, as well as loss of binding materials between grains due to the microbial growth was observed. In addition, small fissures and cavities were formed within stone granules. EDX microanalysis of stone samples revealed that, the control consisted of calcium (Ca), silicon (Si), sodium (Na), aluminum (Al),
122
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
Fig. 3. FTIR analysis of control and treated limestone cubes. A. Control and treated with clove, cinnamon and thyme. B. Control and treated with cetrimonium, PCMC: para chloro meta cresol and TEAB: tetra ethyl ammonium bromide.
Fig. 4. Biodeterioration signs of limestone cubes. A. Inoculated limestone cube (control). B. Turbidity due to microbial activity. C. and D. pigmentation due to Bacillus safensis and Streptomyces rochei growth, respectively.
carbon (C) and (O) oxygen, meanwhile, analysis of infected samples by both strains demonstrated reduction of calcium weight which is the main constituent of limestone. On contrary, sodium and chlorine increased and also new elements appeared like iron and sulfur element suggesting new salt formation. 3.9. Simulation of treatment and conservation processes
Fig. 5. Reduction percentages in the stones physical characteristics caused by Bacillus safensis and Streptomyces rochei growth. Bars represented standard error (n = 3).
Bacillus safensis and Streptomyces rochei were inhibited by cetrimonium (1 mg/mL) and TEAB (0.6 mg/mL), while same amount of cinnamon oil inhibited 99.7% and 99.8% of the two isolates, respectively, when used as treatment after infection. A similar pattern was observed for conservation assessment as, cetrimonium (1 mg/mL) and TEAB (0.6 mg/mL) inhibited 99.6% and 98.9% of Bacillus safensis and Streptomyces rochei, respectively. Cinnamon oil (1 and 5 mg/mL) inhibited 99.1 and 93.1 of the isolates, respectively (Fig. 7).
Fig. 6. ESEM imaging of artificial inoculated limestone. A. Non-colonized stone (control). B. Stone inoculated with Bacillus safensis. C. Stone inoculated with Streptomyces rochei.
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
123
Fig. 7. Simulation of treatment and conserved processes of limestones inoculated with Bacillus safensis and Streptomyces rochei. (1) Cetrimonium against B. safensis inoculated stone; (2) Cinnamon oil against B. safensis inoculated stone; (3) TEAB (tetra ethyl ammonium bromide) against S. rochei inoculated stone and (4) Cinnamon against S. rochei inoculated stone. Bars represented standard error (n = 3).
4. Discussion Limestone has been used as a building material for many centuries in Egypt, representing the cultural heritage of mankind of each period. This study sheds light on the bacterial degradation at 7 different Egyptian archeological sites (upper and lower Egypt), as well as on the methods of treatment and conservation. Lower and Upper Egypt temperature and relative humidity are varied throughout the year consequently, produced different types of microbial infection [26]. Microbial counts of the studied sites revealed that bacteria were predominant over actinobacteria all except at Giza pyramid complex site, where actinobacteria was predominant. This was agreed with Eric et al [27] and Grigorevski-Limaa et al [28] who reported one fold increase in bacteria compared with actinobacteria in different touristic environments. Higher count of actinobacteria at Giza pyramid region was attributed to formation of spores that resist desiccation and starvation for long periods [29]. Previous results found that actinobacterial isolates were able to grow on calcium carbonate mineral medium with slightly alkaline medium (pH 7.8-8), which resemble the natural limestone [30]. The high salt tolerance of actinobacteria qualified them to persist and attack calcium carbonate consequently damaging stones [31]. High percentages of bacterial isolates that were able to grow on mineral medium (75, 66.6% and 55% at Giza pyramid complex, Roman amphitheater and Seti tomb, respectively) were due to low organic contaminants [32]. Other sites including Mosque of Judge Abd El Basset and Senusret I obelisk demonstrated low percentage of stone colonizing bacterial isolates. Mosque of Judge Abd El Basset, on the other hand, was a residential area which is highly polluted and had great sewage drainage problem as well as Senusret I obelisk which is polluted with garbage and fossil fuel [33]. Previous reports demonstrated that, numerous heterotrophic microorganisms present on the stonework can utilize, for their growth, the airborne organic pollutant settled on the stone surface and incomplete combustion of fossil fuel [5]. Acid and pigments production were very common mechanisms in the destruction of arts, where color changes to rock monuments are a real threat to cultural heritage, which is irreversible process [34]. In this study, both bacteria and actinobacteria were found to be acid producers, causing pitting, dissolution and powdering of
stone. Some actinobacterial isolates obtained from the sites under study were able to produce pigments, where no bacterial isolate was a melanin producer [35]. Most of bacterial isolates colonizing deteriorated stone monuments belonged to Gram positive bacteria represented by Bacillus spp. and Streptomyces spp, while Gram negative bacteria had low occurrence. This was in agreement with the previous study of AbdElkareem and Mohamed [36]. In addition, it was reported that the isolated carbonate-dissolving bacteria of limestone were affiliated to families Bacillaceae, Staphylococcaceae as well as Streptomyces species [37]. The most degrading bacterial isolate identified in this study, Bacillus safensis, was also previously recorded as contaminant on fresco which is a technique of mural painting (any piece of artwork painted or applied directly on a wall, ceiling or other surface) [38]. B. safensis is a Gram-positive, aerobic, mesophilic, chemoheterotrophic bacterium [39]. Satomi [40] and Kothari [41] have also reported the isolation of B. safensis strains from environments characterized with conditions like low moisture, high soil salinity, nutrient deficiency, high ultraviolet radiation and temperature, and strong winds, which are similar conditions to Seti tomb at Luxor and Giza pyramid complex site. Streptomyces rochei was considered as mesophilic, alkaliphilic, moderate salt tolerant in nature and strong acid producer from different carbon sources; all such characters enable it to grow within limestone monuments [42]. The present study was extended to determine the efficiency of some chemical and natural products against stone colonizing bacteria. Chemical (PCMC, TEAB and cetrimonium) and natural (cinnamon, clove, pepper mint, lavender, camphor and thyme) compounds were tested for antimicrobial activity. TEAB and cetrimonium were considered as the most common quaternary ammonium compounds in this regard, where they affect the cytoplasmic membrane and cause denaturation of proteins, resulting in leakage of intracellular components and death of bacteria [43]. PCMC was classified as a halo phenolic compound that has both halogen and hydroxyl groups that generally considered cellular poisons, reacting with a cell membrane and variety of macromolecules and microbial structures and inhibits fatty acid synthesis via their reactive hydroxyl and halogen groups [44]. Synthetic antimicrobial agents in this study such as PCMC, TEAB and cetrimonium inhibited the microbial growth at very low con-
124
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125
centrations reached 0.03% (0.3 mg/mL), which was supported by earlier observations [45,46]. Essential oils including thymol and its isomer carvacrol, cinnamon and clove have been used throughout history as preservatives. Oregano and thyme essential oils have been shown to be strongly microbicidal against Gram positive and Gram negative bacteria as well as actinobacteria [47] via destabilizing the cytoplasmic membrane and acting as a proton exchanger [48]. Some studies [49,50] suggested that compounds penetrate the cell, where they interfere with cellular metabolism and phenols such as carvacrol and eugenol (main component of essential oil), disturb the cellular membrane and react with active sites of essential enzymes. Both thyme and cinnamon released high levels of carvacrol, thymol and cinnamaldehyde, respectively, during the first 3–6 h of incubation leading to bacterial inhibition [51]. Concerning natural antimicrobial compounds, cinnamon, clove and thyme have antibacterial activity at very low concentration (0.5% or 5 mg/mL) against almost rock colonizing bacteria, a study which agreed with that of Mironescu et al. [52]. FTIR confirmed safety of the tested antimicrobial compounds to stone material, since no variation or changes in stone chemical composition were observed on the simulated treated stone cubes with the effective MICs of both natural and chemical compounds. Consequently, they could be directly applied at any archeological sites as common antimicrobial compounds. Visual observation of deterioration sings at simulation experiment was agreed with Styriakova et al. [53] who recorded that organic acids production, bacterial colonization and the destruction of the mineral surfaces of grains greatly accelerated the release of mineral elements from rocks to solution, which appeared as turbidity and stone bleaching, although these processes take many years in nature. Dark pigmentation and stone discoloration was clearly obvious on stone surface, these observations were recorded by actinobacteria which tolerated high salt environment [54]. Melanin negative Streptomyces spp. produced different types of diffusible pigment (yellow to dark brown) affecting stone appearance [55]. The deterioration is a complex process that changes the mechanical properties and mineralogical composition of limestone. Concurrently, when gypsum or crust was formed in the pores of limestone it can serve as pore blocking cement [56], and thus it helps in the formation of a non-porous surface crust on the limestone, decreasing the porosity of stone and causes weakening of the host rock [57]. This explained the compressive strength and porosity calculations recorded in this study. Application of synthetic antimicrobial compounds show more bactericidal activities than that of natural one, which may due to high stability and penetration within stone surface especially when they used at control or conservation experiment. This also may be attributed to the high volatility of different essential oils causing smaller residual effect of them inside the stone which was affected by different conditions like oil concentration, texture and porosity of the stone, crust formation, aeration and temperature [58].
5. Conclusion In this study, the microbial load of deteriorated limestone monuments from different Egyptian archeological sites was recorded. The most potential biodeteriogens were identified as Bacillus safensis and Streptomyces rochei. Natural; cinnamon, clove, thyme and synthetic; PCMC, TEAB, certrimonium antimicrobial agents were able to treat and protect stones from the potent stone degrading isolates which could be applicable on archeological sites. Essential oils are effective, safe, available, and cheap which have no residual effect on stone which qualify them to be applied on archeological limestone objects.
Funding source This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1] D.D. Klemm, R. Klemm, The building stones of ancient Egypt - a gift of its geology, J. Afr. Earth Sci. 33 (2001) 63–642. [2] M.B.O. Ortega, C.C. Gaylarde, G.E. Englert, P.M. Gaylarde, Analysis of saltcontaining biofilms on limestone buildings of the Mayan culture at Edzna, Mexico, Geomicrobiol. J. 22 (2005) 261–268. [3] L.K. Herrera, C. Arroyave, P. Guiamet, S.G. Saravia, H. Videla, Biodeterioration of peridotite and other constructional materials in a building of the Colombian cultural heritage, Int. Biodeter. Biodegrad. 54 (2004) 135–141. [4] J.C. Saiz, The role of microorganisms in the removal of pollutants deposited onto historic buildings, Int. Biodeterior. Biodegrad. 40 (1997) 225–232. [5] E. Zanardini, P. Abbruscato, N. Ghedini, M. Realini, C. Sorlini, Influence of atmospheric pollutants on biodeterioration of stone, Int. Biodeterior. Biodegrad. 45 (2000) 35–42. [6] T. Warscheid, J. Braams, Biodeterioration of stone: a review, Int. Biodeterior. Biodegrad. 46 (2000) 343–368. [7] S. Scheerer, M.O. Ortega, C. Gaylarde, Microbial deterioration of stone monuments, Adv. Appl. Microbiol. 66 (2009) 97–139. ˜ [8] G. Pinar, C. Ramos, S. Rölleke, G.C. Schabereiter, D. Vybiral, W. Lubitz, E.B. Denner, Detection of indigenous Halobacillus populations in damaged ancient wall paintings and building materials: molecular monitoring and cultivation, Appl. Environ. Microbiol. 67 (10) (2001) 4891–4895. [9] L. Laiz, D. Recio, B. Hermosin, C. Saiz-Jimenez, Microbial communities in salt efflorescences, in: O. Ciferrio, P. Tiene, G. Mastromei (Eds.), On microbes and art, Proceedings of an international conference on microbiology and conservation, Kluwer Academic/Plenum Publishers, NewYork, 2000, pp. 77–88. [10] J.J. Walker, N.R. Pace, Endolithic microbial ecosystems, Annu. Rev. Microbiol. 61 (2007) 331–347. [11] M.E. Abdel-Haliem, A.A. Sakr, M.F. Ali, M.F. Ghaly, C. Sohlenkamp, Characterization of Streptomyces isolates causing colour changes of mural paintings in ancient Egyptian tombs, Microbiol. Res. 168 (7) (2013) 428–437. [12] E. Küster, Outline of a comparative study of criteria used in characterization of the actinomycetes, Int. Bull. Bact. Nomen. Taxon. 9 (1959) 97–104. [13] Y. Nuhoglu, E. Oguz, H. Uslu, A. Ozbek, B. Ipekoglu, I. Ocak, I. Hasenekoglu, The accelerating effects of the microorganisms on biodeterioration of stone monuments under air pollution and continental-cold climatic conditions in Erzurum, Turkey, Sci. Total Environ. 364 (2006) 272–283. [14] S. Evan, W. Ryan, M. Hasen, A. Schneegurt, Isolation and characterization of halotolerant soil fungi from the great salt plains of Oklahoma, Mycologia 34 (4) (2013) 329–341. [15] W. Song, N. Ogawa, T. Hatta, C.T. Oguchi, Y. Matsukura, Effect of Bacillus subtilis on granite weathering: a laboratory experiment, Catena, Biogeomicrobiol. 70 (3) (2007) 275–281. [16] W.W. Scott, Standard methods of chemical analysis, Fifth edition. Edited by N. Howell Furman, Ph.D. 2 vols, The Technical Press, Ltd.; New York. Price 6d, (1969) pp. 2617. [17] R. Atlas, Alphabetical listing of media, Handbook of microbiological media, 3rd Ed., CRC Press, Inc., Boca Raton, 2004. [18] M. Kawato, R. Shinobu, On Streptomyces herbaricolour, nov sp. supplement: a simple technique for the microscopic observation. Memoirs of the Osaka University of Liberal Arts and Education, B. Nat. Sci 8 (1960) 114–119. [19] H. Heuer, M. Krsek, P. Baker, K. Smalla, E.M. Wellington, Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients, App. Environ. Microbiol. 63 (8) (1997) 3233–3241. [20] S.F. Altschul, T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (17) (1997) 3389–3402. [21] M. Zyani, D. Mortabit, S. El Abed, A. Remmal, S.I. Koraichi, Antifungal activity of five plant essential oils against wood decay fungi isolated from an old house at the Medina of Fez, Int. Res. J. Microbiol. 2 (3) (2011) 104–108. [22] P. Bhatnagar, S.K. Jain, Antimicrobial activity of plant extract against fungi associated with monument deterioration of Gwalior Fort in India, European Academic Res. 2 (5) (2014) 6199–6210. [23] M.R. Derrick, C.S. Dusan, J.M. Landry, Infrared spectroscopy in conservation science, the Getty Conservation Institute, Los Angeles (1999). [24] J. Slusarek, The correlation of structure porosity and compressive strength of hardening cement material, Archit. Civil Eng. Environ. 3 (2010) 85–92. [25] G.D. Danilatos, V.N.E. Robinson, Principles of scanning electron microscopy at high specimen pressures, Scanning 2 (1979) 72–82. [26] M. Domroes, A. El-Tantawi, Recent temporal and spatial temperature changes in Egypt, Int. J. Climatol. 25 (2005) 51–63. [27] E. May, S. Papida, H. Abdulla, S. Taylerl, A. Dewedar, Comparative studies of communities on stone monuments in temperate and semi-arid climate, in: O. Ciferri, P. Tiano, G. Mastromei (Eds.), Of microbes and art: the role of microbial communities in the degradation and protection of cultural heritage, Kluwer Academic Publishers, New York, 2000, pp. 49–62.
K.Z. ElBaghdady et al. / Journal of Cultural Heritage 38 (2019) 118–125 [28] A.L. Grigorevski-Limaa, R.G. Silva-Filhob, L.F. Linharesa, R.R.R. Coelhoa, Occurrence of actinomycetes in indoor air in Rio de Janeiro, Brazil. Build. Environ. 41 (11) (2006) 1540–1543. [29] E. May, Microbes on building stone: for good or ill? Culture 24 (2) (2003) 5–8. [30] R. Ghorbani, R. Greiner, H.A. Alikhani, J. Hamedi, B. Yakhchali, Distribution of actinomycetes in different soil ecosystems and effect of media composition on extracellular phosphatase activity, J. Soil Sci. Plant Nutr. 13 (1) (2013) 223–236. [31] H. Aria, On microorganisms in the tomb of Nefertari, Sci. Conser. 27 (1988) 19–20. [32] L.M. Suihko, L.H. Alakomi, A.A. Gorbushina, I. Fortune, M Saarela, Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments, Syst. Appl. Microbiol. 30 (2007) 494–508. [33] A.A. Ahmed, Land use change and deterioration of pharaonic of monuments in Upper Egypt, J. Eng. Sci. 37 (1) (2009) 161–177. [34] C. Giacobini, M.A. Decicco, I. Tiglie, G. Accardo, Actinomycetes and biodeterioration in the field of fine art, Biodeterioration 7 (1988) 418–427. [35] W.E. Krumbein, Colour changes of building stones and their direct and indirect biological causes, in: R. Delgado, F. Henriques, T. Jeremias (Eds.), Proceedings of the 7th International Congress on Deterioration and Conservation of Stone, Laboratorio Nacional de Engenharia Civil, Lisbon, Portugal, 1992, pp. 443–452. [36] E.A. Abd-Elkareem, R.M. Mohamed, Microbial deterioration of limestone of Sultan Hassan mosque, Cairo- Egypt and suggested treatment, Int. J. Chem. Tech. Res. 10 (5) (2017) 535–552. [37] G. Subrahmaniyam, R. Vaghela, N.P. Bhatt, G. Archana, Carbonate-dissolving bacteria from ‘Miliolite’, a bioclastic limestone, from Gopnath, Gujarat, Western India, Microbes Environ 27 (3) (2012) 334–337. [38] K.M. Reza, N. Ashrafalsadat, R.M. Reza, N. Taher, N. Ali, Isolation and molecular identification of extracellular lipase-producing Bacillus species from soil, Annals Biol, Res. 5 (2014) 132–139. [39] C.E. Raja, K. Omine, Arsenic, boron and salt resistant Bacillus safensis MS11 isolated from Mongolia desert soil, Afr. J. Biotechnol. 11 (2012) 2267–2275. [40] M. Satomi, T. Myron, L. Duc, K. Venkateswaran, Bacillus safensis sp. nov., isolated from spacecraft and assembly facility surfaces, Int. J. Sys, Evol. Microbiol. 56 (2006) 1735–1740. [41] V.V. Kothari, R.K. Kothari, C.R. Kothari, V.D. Bhatt, N.M. Nathani, P.G. Koringa, C.G. Joshi, B.R.M. Vyas, Genome sequence of salt-tolerant Bacillus safensis strain VK, isolated from saline desert area of Gujarat, India, Genome Announc. 1 (5) (2013) e00671–13. [42] R.N. Gopi, D.P.N. Ramakrishna, S.V. Rajagopal, Optimization of culture conditions of Streptomyces rochei (MTCC 10109) for the production of antimicrobial metabolites, Egypt. J. Biol. 13 (2011) 21–29.
125
[43] R. Xuehong, L. Jie, Smart anti-microbial composite coatings for textiles and plastics in: Smart composite coatings and membranes, Woodhead Publishing Ser. Composites Sci Eng (2016) 235–259. [44] G. McDonnell, Sterilization and Disinfection, Encyclopedia of Microbiology (Third Edition) (2009) 529–548. [45] S. Burt, Essential oils: their antibacterial properties and potential applications in foods a review, Int. J. Food Microbiol. 94 (3) (2004) 223–253. [46] N. Matan, H. Rimkeeree, A.J. Mawson, P. Chompreeda, V. Haruthaithanasan, M. Parker, Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions, Int. J. Food Microbiol. 107 (2) (2006) 180–185. [47] T. Nakatsu, A.L. Lupo, J.W. Chinn, R.K.L. Kang, Biological activity of essential oils and their constituents, Studies Nat. Prod. Chem 21 (2000) 571–631. [48] A. Ultee, E.P.W. Kets, M. Alberda, F.A. Hoekstra, E.J. Smid, Adaptation of the food-borne pathogen Bacillus cereus to carvacrol, Arch. Microbiol. 174 (2000) 233–238. [49] L.S. Ooi, S.L. Kam, H. Wang, E.Y. Wong, V.E. Ooi, Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume, Am. J. Chin. Med. 34 (3) (2006) 511–522. [50] P. López, C. Sánchez, R. Batlle, C. Nerín, Development of flexible antimicrobial films using essential oils as active agents, J. Agric. Food Chem. 55 (21) (2007) 8814–8824. [51] E. Pinto, L. Vale-Silva, C. Cavaleiro, L. Salgueiro, Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species, J. Med. Microbiol. 58 (11) (2009) 1454–1462. [52] M. Mironescu, C. Georgescu, L. Oprean, Comparative sporicidal effects of volatile oils, J. Agroalimentary Process. Technol. 15 (3) (2009) 361–365. [53] I. Styriakova, I. Styriak, H. Oberhansli, Rock weathering by indigenous heterotrophic bacteria of Bacillus spp. at different temperature: a laboratory experiment, Mineral. Petrol. 105 (2012) 135–144. [54] H. Abdulla, E. May, M. Bahgat, A. Dewedar, Characterisation of actinomycetes isolated from ancient stone and their potential for deterioration, Pol. J. Microbiol. 57 (3) (2008) 213–220. [55] M. Oskay, Antifungal and antibacterial compounds from Streptomyces strains, Afr. J. Biotechnol. 8 (13) (2009) 3007–3017. [56] Á. Torok, Surface strength and mineralogy of weathering crusts on limestone buildings in Budapest, Build. Environ. 38 (9) (2003) 1185–1192. [57] Á. Torok, R. Rozgonyi, Mineralogy and morphology of salt crusts on porous limestone in urban environment, Environ. Geol. 46 (3) (2004) 323–339. [58] M.E. Young, H.L. Alakomi, I. Fortune, A.A. Gorbushina, W.E.I Krumbein, C. McCullagh, P. Robertson, M. Saarela, J. Valero, M. Vendrell, Development of a biocidal treatment regime to inhibit biological growths on cultural heritage, Environ. Geol. 56 (2008) 631–641.