Bacterial Deterioration in the Limestone Minaret of Prince Muhammad and Suggested Treatment Methods, Akhmim, Egypt

El-Amir Muhammad’s minaret in Akhmim, Sohag, Egypt, is constructed of limestone and has been exposed to many factors of damage as a result of the high levels of ground water. Limestone is strongly affected by ground water, especially when being impure. The current work discusses the results of ana-lytical techniques including chemical testing to determine the types of soluble salts through optical microscopy, electronic scanning electron microscope with an X-ray energy dispersion system (ESEM) to study and determine the causes of rapid degradation. Microbial weathering phenomena toward limestone were also studied. Different bacteria and fungi were isolated from outdoors and indoors of air and limestone of the building of which Bacillus cereus OK447647, B. subtilis OK447648, Serratia marcescens OK447650, Pseu-domonasoryzihabitans OK447649, Aspergillus flavus, A. niger, Penicillium chrysogenum and Cladosporium cladosporoids were the most representative. B. cereus OK447647 and B. subtilis OK447648 have shown ability for calcium carbonate dissolving. The minimal inhibitory concentrations (MICs) of sodium azide were investigated against the growth of microbial isolates. Sodium azide at 100 ppm was found to be the best treatment for bacterial isolates al-though it had no significant effect against fungi.


Introduction
The mosque and minaret of El-Amir Muhammad dates to the Ottoman tech-nology and is known as the Mosque of the Market. It was constructed by the aid of El-Amir Muhammad, El-Amir Hassan's father. This minaret is the most effective one left in the mosque. It is located at the western aspect of Caesarea Street ( Figure 1).
The minaret consists of three floors. The first floor has a rectangular projection, the second is an octagonal projection, and is cylindrical in shape, crowned with the aid of the pinnacle of the minaret, that's punctuated with the aid of using six knotted holes and the third is cylindrical in shape and is topped by the top of the minaret punctuated by six knotted slots. Many researchers have discussed the damage to archaeological Islamic minarets [1] [2] [3] [4]. Limestone has centuries-lengthy and global culture as a constructing material [5] [6] [7].
Solution weathering is continual but generally unthreatening to the structure of a building. One such mechanism is salt weathering. Salts occur naturally within the atmosphere e.g. at the coast, but in polluted environments, their concentrations and variety are increased because of the chemical reactions between limestone and acid pollutants [8]. The most ordinarily salt produced by this reaction is calcium sulphate (gypsum) which is the most often salt related to weathered limestone [9] [10]. On drought, the salt crystals precipitate either on the surface, or within the pores of the stone. This can cause dislodgement of individual grains (granular disintegration) or the event of scales and flakes of stone [11] [12]. Salt weathering is most cases natural stone decay, and as a consequence, there is a significant problem with the conservation of cultural heritage [13].
Dissolved salts such as, sulfate, chloride, sodium nitrate, potassium, magnesium, ammonium and calcium are major factors in the damage of porous materials such as limestone. The rate of spoilage is also attributed to the behavior of salt with limestone, leading to deep crushing, partial and surface cracking, grain damage and pits as well as physical stress as a result of crystallization, hydration and differential thermal expansion.
The biodeterioration of monumental heritage is a worldwide phenomenon; it represents a significant loss of cultural heritage [14]. Microbial biodeterioration  is one of the main causitive of archeological rocks deterioration especially in museums and mosques [15] [16]. Stone surfaces and paintings of monuments are exposed to continuous biodegradation and biodeterioration agents, such as microbial communities colonizing stone surface and paintings consisting of a great number and diversity of microorganisms, such as bacteria, action bacteria, fungi, and yeast [17].
The biodeterioration of archeological stones works occurs as a consequence of biofilm production, secretion and deposition of organic and inorganic compounds and physical penetration of microbes [18] [19] Figure 2(e) and Figure  2(f). The growth and activity of the microorganisms on stone surface results in major alterations such as surface alterations (etching, pitting, stratification, etc), staining or color alteration, bio-weathering (stone dissolution), bio corrosion and transformation of crystal into small size one [20]. The monitoring of microbial contamination represents the basis for a proper conservation strategy [21].
The present work aimed to investigate and identify the biological cause of archeological limestone biodeterioration from El-Amir Muhammad's minaret in Akhmim-Egypt and suggest methods of treatment. Figure 2. Effect of moisture on the lower walls (a), discoloration of limestone (b), the presence of separation and loss of some stone blocks ((c), (d)) and the effect of microbiological damage on the walls ((e), (f)) of El-Amir Muhammad's minaret.

Collection of Limestone Samples
Limestone samples were collected from the study site (El-Amir Muhammad's Minaret, Akhmim, Sohag, Egypt) by non-destructive methods.

Petrographic Examination
Nikon polarizing microscope (JEOL JSM5500LV) was used in the petrographic study of limestone samples.

Scanning Electron Microscope (SEM)
SEM was used to study and understand the fine structure, decomposition, and different properties of the limestone under study. It was carried out in the Central Lab, South Valley Univ. using JEOLJSM-5500 LV SEM (JEOL, Japan).

Isolation of Airborne Microorganisms
The settle plate method [22] was used to estimate the airborne spores in of El-Amir Muhammad's minaret. Nutrient agar and Czapek's (CZ) agar media were used for isolation of bacteria and fungi, respectively. The plates were exposed for five minutes. Nutrient agar plates were incubated at 37˚C for 72 h while CZ plates were incubated at 28˚C for 7 days. The developed colonies were counted in plates and the average number of colonies per three plates was determined.

Isolation of Microorganisms from Deteriorated Limestone
Dry cotton swabs were rubbed on the surface of the deteriorated parts of the building over an area of 4 cm 2 , under aseptic conditions, stored at 4˚C until used for inoculation as mentioned previously.

Identification of Bacterial Isolates
The bacterial isolates were tentatively identified on the basis of classification schemes published in Bergey's Manual of Systematic Bacteriology [23].

Molecular Identification of the Common Bacterial Isolates
The Bacterial isolates were cultured in sterile test tubes containing 10 ml of nutrient broth medium [24]. The cultures was incubated at 28˚C for 48 hours, then sent to the molecular Biology Research Unit, Assiut University for DNA extraction using Patho-gene-spin DNA/RNA extraction kit provided by Intron Biotechnology Company, Korea. From each sample the DNA was sent to SolGent Company, Daejeon South Korea for polymerase chain reaction (PCR) and gene sequencing. PCR was performed using two universal primers where 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used. The purified PCR products (amplicons) were reconfirmed using a size nucleotide marker (100 base pairs) by electrophoreses on 1% agarose gel. The amplicons were sequenced with the incorporation of dideoxynucleotides (dd NTPs) in the reaction mixture. Bacterial amplicons were sequenced in the sense and antisense directions using 27F and 1492R primers [25]. Sequences were further analyzed using Basic Local Align-  [26] and optimized manually.
The positions where one or more species contained a length mutation and ambiguously aligned regions were not included in the subsequent phylogenetic analysis. Maximum parsimony and maximum likelihood analyses were made in PAUP 4 [27]. Maximum-parsimony (MP) trees were obtained by 100 random addition heuristic search replicates, and 1000 bootstrap replicates were performed employing 5 random addition heuristic searches. Maximum-likelihood (ML) analysis [28] was performed using heuristic searches with the random stepwise addition of 100 replicates and tree bisection-reconnection (TBR) rearrangements. The optimal model of nucleotide substitution for the ML analyses was determined using hierarchical likelihood ratio tests (hLRTs) Model test 3.7 [29]. The model selected as the best fit for 16s rDNA dataset was TrN. Phylogenetic trees were visualized using Njplot [30] and edited in Adobe Illustrator CS6.

Determination of the Minimal Inhibitory Concentration (MIC) of (Sodium Azide) against Isolated Microorganisms
The microcide solution was prepared by dissolving sodium azide ethyl alcohol 95% to give concentrations ranging from 50 up to 150 µg/l (ppm). The most dominant bacterial and fungal isolates were treated by different concentrations (25,50, 100 and 150) ppm of sodium azide using the agar well diffusion method [35]. Control without microcide was done using ethyl alcohol. Inhibition zones were measured to determine the minimum inhibition concentration

Weathering of Limestone
In the present case, limestone in general is quite homogeneous in its chemical  [37]. Limestone, which is often used in stone construction, where the effects of sulfate attack appear very quickly, especially in cases of stone damage, the formation of gypsum crusts, results in stone cracking [38] (Figure 2(b) and Figure 2(c)). Humidity increases the chances of damage due to wet and dry thawing and freezing. Where the soluble salts move in and out of the components of the porous stones. Seasonal changes in soil and temperature also control, which leads to the appearance of salt flowers that are soluble (Figure 2(a)). Weathering is also related to mechanical strength, water absorption and permeability of the stone and treatment work must include the estimation and improvement of these properties. The pore space in the weathering zone of limestone becomes clogged when the process of recrystallization of the salts takes place as a result of damage to its components and cause discoloration of limestone [39] ( Figure 2(b)). Water has an important role in the damage of limestone because it is highly polar [40]. It also helps in the rate of deterioration of limestone, the high content of clay minerals, which are expandable minerals. The extent of the ability of limestone to absorb moisture is expressed in estimating the clay minerals content, as water retention is a result of the presence of clay minerals in the pores. The presence of clay minerals in the formation of limestone in addition to the characteristics of limestone such as its high porosity causes severe damage to limestone caused separation and loss of some stone blocks (Figure 2(c) and Figure  2(d)). Many cracks were observed due to swelling and contraction of the components of the clay minerals due to changes in relative humidity, and the expansion and contraction of the degraded limestone components as a result of the changing temperature cycles. The degree and depth of damage also indicates that the components of the minaret have been damaged by water due to previous treatments, as they appear as visible white sediments caused by microbiological damage on the stone surface Figure 2(e) and Figure 2(f). Optical microscopy revealed some details such as porosity, fine cracks and grain morphology, the grains formed a fragmented matrix of heterogeneous, non-solid powders, with a large amount of fine cracks. The study showed that the limestone grains contain skeletal and fossil parts of fossils of the foraminifera, especially the nemolite, which consist of calcium carbonate CaCO 3 and dolomite, were found to fill the cracks and dissolve with some traces of gypsum CaSO 4 •2H 2 O. Note calcite sparite in the inner spaces of the fossils, and the main mineral is micritic calcite as shown in Figure 3.
XRD results showed that calcite (CaCO 3 ) is the main component of limestone samples, and the presence of CuFeNaS 2 compound is an indicator of the presence of gypsum and halite with gypsum (CaSO 4 •2H 2 O) ( Figure 4).
Unexpectedly, XRD analyzes failed to determine the presence of aluminum silicate or clay minerals, as this was inconsistent with the results obtained from ESEM-EDS analyzes, where the latter was able to detect aluminum silicate within the polished samples and this may be due to the presence of compounds Clay

Identification of the Bacterial Isolates
Based on morphological, physicochemical and physiological characterization (

Phylogenetic Analyses of Common Bacterial Strains
The   and P. oleovorans with high statistical support (93/89/100 for MP/ML/BYPP, respectively) and the fourth strain RM was grouped with several strains of Serratia marcescens (Figure 7).

Estimation of Airborne Microorganisms
Six airborne bacterial genera: Bacillus, Staphylococcus, Micrococcus, Streptomyces Pseudomonas and Serratia were recovered from out and indoor of El-Amir Muhammad's minaret. The data in Table 3 show what Bacillus cereus OK447647 and B. subtilis OK447648 were the most common and the most frequent as well comprising (53.9%; 43.3% and 30.5%; 33.9%) of total counts from outdoor sand indoors, respectively. Gram negative bacteria were identified as Pseudomonas oryzihabitans OK447649 and Serratia marcescens OK447650 comprising (0.4%; 0.3% and 0.9%; 0.5%) of total counts from outdoor and indoor bacterial isolates, respectively.  Figure 8(a) also reveal that Bacillus cereus was dominant, it was recorded from 100% of samples collected from indoor and outdoor aerosols of El-Amir Muhammad's minaret, followed by Staphylococcus aureus and Streptomyces (60%; 100% and 60%; 60%), Serratia marcescens OK447650 and Pseudomonas oryzihabitans OK447649 were less frequent, in outdoors and indoors, respectively. Gram-negative bacteria were found in low numbers. Table 3 and Figure 8(b) show that eleven airborne fungal species belonging to five genera were recovered from out and indoor of El-Amir Muhammad's minaret. Aspergillus was the most prevalent genus represented by six species of which Aspergillusflavus was dominant it was recovered from 100% of samples comprising (34.2% and 23.8%) of total fungi from outdoors and indoors, respectively. Penicilliumchrysogenum was of moderate occurrence comprising (26.3% and 14.3%) of total fungi from outdoors and indoors, respectively, followed by Cladosporiumcladosporoids representing (10.5% and 22.2%) and was recorded from (100% and 80%)% of total fungi from outdoors and indoors, respectively. Lower frequencies were revealed by other genera as Alternariaand Syncephalustrum.

Data in
Among airborne fungi isolated from outdoor and indoor of El-Amir Muhammad's minaret, Aspergillus flavus, A. niger and Penicillium chrysogenum were the most frequent. The presence of Alternaria alternate and Cladosporium cladosporoids was pointed out. Similar results were reported by Abdel Hameed 2009 [45]; Gillum and Levetin 2008 [46]. Airborne spores and cells may be carried by the wind or by human activities or deposited onto the wall surfaces by gravitational settling [47]. Most microorganisms are able to successfully grow on stone surfaces covered with dust, animal remains, air contaminants and secretion or finger-marks, creating invisible layer of biofilm (Rajendran and Nisy 2012).

Microorganisms from Deteriorated Limestone
According to data in Table 3 and Figure 9 [49] stated that Strains of Bacillus spp. are some of most oftenly found bacteria identified on surface as well as inside the stone artifacts. Furthermore, Micrococcus, Staphylococcus, were recovered from pre-historic rock-paints of Kabra-pahad, India [50]. Table 3 and Figure 9(b) depict that twelve species belonging to seven fungal genera were recovered from outdoor limestone of which Aspergillusniger

Calcium Carbonate-Dissolving G Microorganisms
Microbial metabolites enable some substances from rocks or minerals such as Si, Al, Fe, Mg, Mn, Ca, K, Na, Ti, to leach out from their salts, especially because of the impact of microorganisms on the dissolving rate of minerals [55].
Two bacterial isolates were positively identified as being capable of dissolving calcium carbonate, they belong to the genus Bacillus: B. cereus OK447647 and B. subtilis OK447648 ( Figure 10). These results come in agreement with Abd-

Determination of the Minimal Inhibition Concentration (MIC) of (Sodium Azide) against Isolated Microorganisms
Based on the results shown in Table 4, sodium azide of concentrations up to 50 ppm were ineffective against all tested bacteria and fungi. At 100 ppm, all tested Figure 10. Zone of clearance of carbonate-dissolving Bacillus cereus OK447647 (1), and B. subtilis OK447648 (2) on DB medium. microorganisms were inhibited, the mean diameter of inhibition zone fluctuated between 8 and 15 mm. Therefore, it can be concluded that 100 ppm was the MIC of sodium azide to inhibit all the tested bacteria and fungi. Similar findings were reported by Abdelhafez et al. (2012), who concluded that 100 ppm of sodium azide was the best treatment to stop the growth of all microbial isolates recovered from surfaces of archeological marble located in Cairo, Egypt.

Conclusions
It is clear from the visual examination of the limestone samples under study that they are exposed to large levels of damage with a high percentage of different salts, as evidenced by the presence of granular disintegration and cracks and the presence of salt crystals of different sizes that can be observed with the naked eye. This was confirmed through analyzes, as the work of microscopy, electron microscopy, X-ray fluorescence and X-ray diffraction showed the presence of soluble salts with the presence of aluminum silicate compounds, clay minerals with a high content of calcite as an essential component of the limestone under study, as was confirmed by analyzes X-ray diffraction also observed the presence of fine cracks in the limestone distributed in an unbalanced manner to the grains as a result of the impact of the destructive materials on the calcite. The calcite grains cause further acceleration of the limestone damage processes as a result of the internal disintegration that results from the activity of the salts.
Microorganisms have a destructive impact on El-Amir Muhammad's minaret limestone walls. The presence of calcium carbonate dissolving bacterial species such as B. cereus OK447647 and B. subtilis OK447648 causes severe biodeterioration of the walls. Fungal species such as Aspergillus, Penicillium, Cladosporium and Alternariacan cause many aesthetical damages to stone monuments.
More attention should be paid to salt weathering, soiling, discoloration and changing microflora. The conservation of the heritage monument is a challenging task. To ensure sustainable conservation, treatments have to be safe to the protected object, eco-friendly, derived from a renewable resource and low cost in application.