Scholarly article on topic 'Microbial deterioration of archaeological marble: Identification and treatment'

Microbial deterioration of archaeological marble: Identification and treatment Academic research paper on "Biological sciences"

CC BY-NC-ND
0
0
Share paper
Academic journal
Annals of Agricultural Sciences
OECD Field of science
Keywords
{"Archeological marble" / "Microbial deterioration of marble" / "Physicochemical properties of marble" / "Deteriorating factors"}

Abstract of research paper on Biological sciences, author of scientific article — A.A.M. Abdelhafez, Fatma M. El-Wekeel, E.M. Ramadan, A.A. Abed-Allah

Abstract Microbial deterioration of archeological marble was studied on samples taken from three locations in Cairo, Egypt; Mohamed Ali palace, El-Ghory Mosque and Mosque of El-Kady Abdel-Baset. Sampling resulted in 110 microbial isolates, identified as eight fungal genera, three bacterial genera, one actinomycetes genus and six algae. Isolated strains were all identified up to species. The inhibitory effect of five antimicrobial agents at various concentrations was investigated against the growth and development of these microbial isolates. Sodium azide at 100ppm was found to be the best treatment for both fungal and bacterial isolates. Colored spots, caused by microbial growth, were treated by different synthetic and natural chemical substances. Results showed also that microbial enzymes produced by Aspergillus flavus isolate was the best decolorization treatment.

Academic research paper on topic "Microbial deterioration of archaeological marble: Identification and treatment"

Annals of Agricultural Science (2012) 57(2), 137-144

Faculty of Agriculture, Ain Shams University Annals of Agricultural Science

www.elsevier.com/locate/aoas

Original Article

Microbial deterioration of archaeological marble: Identification and treatment

A.A.M. Abdelhafez a *, Fatma M. El-Wekeel b, E.M. Ramadan a, A.A. Abed-Allah a

Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, 11241 Cairo, Egypt Center of Research and Conservation of Antiques, Supreme Council of Antiques, Cairo, Egypt

Received 11 June 2012; accepted 25 June 2012 Available online 26 October 2012

KEYWORDS

Archeological marble; Microbial deterioration of marble;

Physicochemical properties of marble;

Deteriorating factors

Abstract Microbial deterioration of archeological marble was studied on samples taken from three locations in Cairo, Egypt; Mohamed Ali palace, El-Ghory Mosque and Mosque of El-Kady Abdel-Baset. Sampling resulted in 110 microbial isolates, identified as eight fungal genera, three bacterial genera, one actinomycetes genus and six algae. Isolated strains were all identified up to species. The inhibitory effect of five antimicrobial agents at various concentrations was investigated against the growth and development of these microbial isolates. Sodium azide at 100 ppm was found to be the best treatment for both fungal and bacterial isolates. Colored spots, caused by microbial growth, were treated by different synthetic and natural chemical substances. Results showed also that micro-bial enzymes produced by Aspergillus flavus isolate was the best decolorization treatment. © 2012 Faculty of Agriculture, Ain Shams University. Production and hosting by Elsevier B.V. All rights

reserved.

Introduction

Marble and limestone are two of the most commonly encountered materials in historic structures. Marble is a metamor-phism of limestone as a result of pressure and heat in the earth crust due to geological processes. Marble has numerous applications as building material, in monuments, for structural and decorative purposes (Winkler, 2002).

* Corresponding author.

E-mail address: journalaaru@yahoo.com (A.A.M. Abdelhafez). Peer review under responsibility of Faculty of Agriculture, Ain-Shams University.

Both limestone and marble can deteriorate by different causes. Physical, chemical and biological factors are all considered deteriorating factors. One of the main causes of deterioration of archeological rocks, especially in museums and mosques, is the microbial action, or biodeterioration. Stones of art works can be colonized by different groups of microorganisms, i.e. bacteria, cyanobacteria, algae and fungi. Micro-bial populations present in a stone substratum are usually the result of successive colonization by different microorganisms that has taken place over several years (Macedo et al., 2009). Microorganisms damage stone in a variety of ways; some create surface deposits, others cause discoloration, pitting or accelerated weathering. Chang and Li (1998) tested the ability of ectomycorrhizal fungi and their associated microorganisms to weather lime stone, marble and calcium phosphate. Research on biological deterioration of marble was done on bacteria, algae, fungi and lichens. Mosses and liverworts have

0570-1783 © 2012 Faculty of Agriculture, Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aoas.2012.08.007

received comparatively less attention because their impact has been considered primarily esthetic. Inorganic materials have always been good substrates for a large number of different microorganisms. Generally, all types of microorganisms are able to attack and degrade marble materials. Biological deterioration could be due to the excretion of metabolic intermediates and/or end-products as well as exoenzymes.

Fungi are among the most active microorganisms in these processes where they can use organic support as nutrients, whereas in the case of inorganic supports, substances are transformed by several metabolites which are excreted and that may react with the support in different ways. Because of their het-erotrophic nature, biological deterioration by fungi is done by altering these supports by synthesizing certain compounds, such as inorganic and organic acids, to utilize the contents of these supports. Fungi were isolated from numerous instances from antique marble and historical limestone buildings at the Acropolis of Athens, the island of Corfu, and from sites in Germany, Italy, Portugal, Russia, Crimea, Spain and Namibia (Gorbushina et al., 1993).

Among numerous bacterial species isolated from stone, Micrococcus was isolated from various stones and found to adhere to substrates and produce exopolysaccharide substances (Urzi et al., 1991). Halo-alkalophilic and methylotrophic bacteria were isolated from deteriorating marble in the Moscow Kremlin masonry and found able to utilize methanol, methylamine, tri-methylamine and fructose as carbon and energy sources (Doronina et al., 2005). Cyanbacteria and Cyanophi-lous Lichens were also found on deteriorating limestones in Jerusalem and Rome (Danin and Caneva, 1990). Dark patches on marble monuments in Rome indicated the presence of Cya-nobacteria. Green microalgae, including Coccomyxa, were found to colonize ornamental marble statues in the Bobli Gardens of Florence (Italy) (Lamenti et al., 2000).

Different types of microcides were used for the treatment of deteriorating marble stone. Costa et al., (1956) studied the efficiency of 18 fungicides for the protection of pine kraft paper against Aspergillusniger, A. repense, A.flavus, Memoniella echi-nata and Rhizopus stolonifer. Growth of Trichoderma koningii was inhibited in the presence of 2 mg/l benomyl while lower concentrations (0.1 and 0.5mg/l) increased fungal biomass through the stimulation of mycelia branching (El-Mougith, 1999).

Mixture of Povidone-iodine and dichloroxylenol was used for the treatment of microbial biodeterioration on limestone inside Tut Ankh Amon tomb (Ammar and El-Deeb, 1992).

The present work aimed to recognize and identify the biological cause of archeological marble biodeterioration in different places in Egypt and suggest methods of treatment.

Materials and methods

Sampling

Samples were collected from antiques showing symptoms of microbial infection, including tables, head of marble statue and mihrab (a semicircular niche in the wall of a mosque) from three locations all in Cairo, Egypt. These locations were Mohamed Ali palace (Shoubra Elkheima area), Al-Ghory Mosque (Elghoria area) and El-Kady Abdel-Baset mosque (Bab El-Shaaria area). Sampling was carried out by two

methods: (a) scratching the surface of infected marble by sterile spatula and transferred right onto three prepared agar media (Dox's agar for fungi, Nutrient agar for bacteria and Starch nitrate agar for actinomycetes). Plates were then incubated at 28-30 0C for 1-7 days, depending on the microorganism and (b) picking by sterilized cotton swabs from the surface of infected marble under aseptic conditions, kept in sterile bag until used, immediately, for inoculation as mentioned above.

Identification of microbial isolates

Identification of bacteria

Plates of nutrient agar medium were inoculated by each sample and incubated for 3 days at 28 0C. The grown colonies were purified on the same medium and after incubation, each single colony was picked up and purified for identification according to Bergey's Manual of Systematic Bacteriology (Holt, 1986; Stanley et al., 1989) and Cowan and Steels, 1974) using biochemical tests.

Identification of actinomycetes

Samples were inoculated on starch nitrate agar medium and incubated for 7 days at 28 0C. Actinomycetes were identified by the same method as in bacteria except that for growth medium. Actinomycetes colonies were characterized according to Holt (1986) and Cowan and Steels (1974).

Identification of fung

Plates of Dox's agar medium were inoculated with each sample and incubated for 1-7 days at 28 oc. The grown colonies were purified on the same medium and the incubated; each single colony was picked for identification according to Gilman (1974), Domsch etal. (1980a,b) as follows: morphological characteristics were determined with the use of light microscope (CarlZeis Microscope with analysis unit) and compared with the standard criteria present in standard references.

Identification of algae

Pieces of marble, weighing 10 g, were mixed in bottles containing 100 ml of sterilized tap water. Bottles were incubated at ambient temperature under continuous illumination of fluorescent lamp (5000 lux) until algal growth appeared. Algae were identified according to Sze (1998).

Biochemical tests

The following testes were used according to Holt (1986), Cowan and Steels (1974) for identification of bacteria and actinomycetes: Motility, MR and VP tests. Casein, Gelatein, Argenine and Starch hydrolysis. Catalase, Indole and Urease production. Growth at 7% NaCl, at 45 oc, at 65 oc and under anaerobic condition. Sugar fermentation (sucrose, sorbitol, raffinose, glycerol, trehalose, D-glucose, L-arabinose, D-xylose and D-manitol). Nitrate reduction and Citrate utilization.

Determination of minimal inhibitory concentration (MIC) of antimicrobial agents against isolated microorganisms

Five commercially available microcides were obtained from Aldrich Company (Germany) and used for determining their minimal inhibitory concentration against each of the microbial isolates. These microcides were:

• Dichloroxylenol C8H8Cl2.

• Thymol 2-[(CH3)2CH]C6-H3-5-(CH3)OH.

• Penta-chlorophenol C6CL5OH.

• Sodium azide NaN3.

• p-crysol C7H8O.

Stock solutions were prepared by dissolving 1 g of each microcide in 1 l of ethyl alcohol 95% to give a concentration of 1000 ig/ml. Gradient concentrations ranging from 25 to 800 ig/ml were prepared by diluting the stock solution with alcohol to obtain the desired concentration. From each micro-cide concentration, 100 il was placed in the three pores, as replicates, in pre-inoculated plate of nutrient agar for bacteria, starch nitrate for actinomycete or Dox's medium for fungi then incubated at 30 0C for 48 h for bacteria and actinomycete, or for 1-7 days. Inhibition zones were measured to determine the minimum inhibition concentration (MIC) that inhibited all fungal species, according to the method of Brantner et al. (1993). Control without microcide was done using ethyl alcohol. Fungi were inoculated using spore suspension, prepared as described below.

Preparation of fungal spore suspension

Dox's agar plates were inoculated with the tested fungal strain. Maximum sporulation was achieved after 7 days of incubation at 30 0C. Spore suspension was prepared by adding 10 ml of sterilized saline solution (8.5 g NaCl/l H2O) to each plate and then spores were scraped with a brush and used in MIC experiments (mentioned above).

Treatment the infected marble

Treatment of the infected marble specimen was done in two steps; first disinfection of the infected marble and second removing the color resulted from the microbial growth. First, disinfection was done by brushing the marble specimen with the best antimicrobial agent, chosen from the previous step. After 6 months, samples were taken by swabbing the marble and inoculated onto Dox's and nutrient agar plates to determine the microbial survival. In the second step, decolorization of infected marble was applied after 6 months from treatment by brushing the marble with one of following seven compounds twice for 48 h: (1) Acetone (95%), (2) Ethyl alcohol (95%), (3) Hydrogen peroxide (6%), (4) Di-ethyl ether (99%), (5)1,2 -Dioxin (?), (6) Mixture of ammonium carbonate 50 g + EDTA 30 g in 1 l distilled water, and (7) Microbial enzymes produced from Aspergillus flavus prepared as follows: Asp. flavus was cultivated in Dox's mineral broth, supplemented with oil as a carbon source at 10 ml/l concentration, and incubated for 7 days at 30 0C. At the end of incubation period, fungal culture was filtrated and the supernatant was used for cleaning the infected marble specimen (Konkol et al., 2009).

Effect of treatment with the above chemicals on the growth of microbial isolates and removal of the microbial color spots was determined by taking swabs from the surface of marble after complete cleaning and cultivated on the corresponding medium and determining the survival microbial growth.

Results and discussion

Sampling

Taken by swabs, two samples were taken from Mohamed Ali palace, one sample from El-Ghory Mosque in El-Ghoria and one sample from El-Kady Abdel-Baset Mosque. Sampling results showed numbers of fungal, bacterial and actinomycetes colonies, respectively, as follows: 29, 3 and 4 from the first sample, 12, 0 and 0 from the second sample, 40, 2 and 0 colonies from the third sample and 20, 0 and 0 from the fourth sample (Table 1).

Identification of the microbial isolates

The resulted microbial colonies were subjected to preliminary characterization depending on the type of organism. Results showed 101 fungal colonies, which were subjected to preliminary characterization according to morphology (Gilman, 1974) and Domsch etal. (1980a,b). They were identified to belong to the following eight genera: Aspergillus, Penicillium, Acremo-nium, Fusarium, Rhizopus, Cladosporium, Alternaria, and Stachybotrys (Table 2). From these results, it can be seen that genus Aspergillus spp. was the most predominant organism isolated from all swabs of deteriorated marble followed by Acre-monium and Penicillium spp.

Comparable results were obtained by Monte (2003) who reported that the oxalate formation on marble specimens is due to the metabolic activity of strains of Aspergillus and Penicillium sp. Cappitelli et al., (2007) isolated species belonging to the genera Alternaría, Cladosporium, Epicoccum and Aspergil-lus from the surface of infected marble.

Isolates of each genus were subjected to species characterization based on morphological characteristics. Results showed that isolates of Aspergillus were A. niger, A. flavus, A.fumigatus, A. sulphureus, A. oryzae, A. humicola, A. clavatus and A. parasiticus (Table 3).

Isolates of Penicillium were identified as P. stolenferum and P. oxalicum (Table 4) and isolates of Acremonim were identified to be A. murorum, A. kilinese and A. fusidiodes (Table 4). Other isolates were identified as Fusarium oxysporium, Rhizo-pus oryzae, Cldosporium herbarum, Alternaria alternata and Stachybotrys chartartum (Table 5).

Identification of bacterial isolates was done by inoculation of samples onto nutrient agar, resulting in five bacterial isolates. These isolates were characterized according to Cowan and Steels (1974) and Holt (1986) and identified as Streptococcus thermophilus, Bacillus brevis, B. coagulans, and one actinomy-cete (Table 6). Actinomycetes isolates were characterized according to Bergey's Manual (Holt, 1986) and all were identified to be Nocardia asteroide. Comparable results were obtained by Claudio et al., (2009) where they isolated bacteria belonging to the genus Bacillus from untreated painted surfaces in the Chapel of the Holy Nail in the medieval ex-hospital of Siena.

Samples also contained three cyanobacterial isolates identified, according to Sze (1998), to be Anabaena, Calothrix, and Oscillatoria, and three isolates of green algae identified as Chlo-rococcum, Chlamydomonas and Volvox. Similar results were also obtained by Danin and Caneva (1990) who isolated Cyan-bacteria and Cyanophilous lichens from the deterioration of limestone walls in Jerusalem and marble monuments in Rome.

Table 1 Total number of fungal, bacterial and actinomycetes isolates detected in infected marble samples.

No. Sample location Physical observation Total number

Fungi Bacteria Actinomycetes

1 Mohamed Ali palace (Table) Brown spots 29 3 4

Black spots

Roughness, Crust

2 Mohamed Ali palace (Head marble) Brown spots 12 ND ND

3 El-Ghory Mosque Black spots 40 2 ND

Roughness

4 ElKadly Abdel-Baset Mosque Black spots 20 ND ND

Total 101 5 4

ND: no data recorded.

Table 2 Nomination of the detected fungal genera and their isolates' distribution among the collected marble samples.

No. Description Aspergillus Penicillium Acremonim Fusarium Rhizopus Cladosporium Alternaría Stachybotrys

1 Mohamed Ali palace (Table) 23 2 2 2 1 1 ND ND

2 Mohamed Ali palace (Head marble) 8 2 ND ND ND 1 1 ND

3 El-Ghory Mosque 20 3 6 2 1 4 2 2

4 ElKadly Abdel-Baset Mosque 20 ND ND ND ND ND ND ND

Total 71 7 8 2 2 6 3 2

ND: no data recorded.

Table 3 Identification of Aspergillus species, isolated from archeological marble.

No. A. niger A. flavus A. fumigatus A. sulphureus A. oryzae A. humicola A. clavatus A. parasiticus

1 10 9 3 2 1 ND 1 ND

2 5 6 1 ND ND 2 ND 1

3 2 3 3 ND 2 ND 2 ND

4 5 4 3 2 1 1 ND 2

Total 22 22 10 4 4 3 3 3

ND: no data recorded.

Table 4 Identification of Penicillium and Acremonium species, isolated from archeological marble.

No. P. Stolenferum P. oxalicum A. murorum A. kilinese A. fusidiodes

1 2 2 1 1 ND

2 ND ND ND ND 1

3 1 ND 2 ND 2

4 ND 2 ND 1 ND

Total 3 4 3 2 3

ND: no data recorded.

Determination of minimal inhibitory concentration (MIC) of antimicrobial agents against isolated microorganisms

Against fungal isolates

This experiment was designed to test the effect of five antimicrobial compounds; Dichloroxylenol, Thymol and para-cresol, at concentrations from 50 to 800 ppm, and penta-cholorophenol and Sodium azide, at concentrations from 25 to 500 ppm, on the isolated fungal strains.

Results of fungi experiment are shown in (Table 7). Dichlo-roxylenol microcide, at concentrations up to 200 ppm, had no effect on fungal growth. At 300 ppm, only Aspergillaus parasiticus was inhibited, while 400 ppmm inhibited Asp. Parasiticus, Fusarium oxysporium and Stachybotrys chartarum, and most were inhibited at 500 ppm. Only at 600 ppm, all of the isolated fungal strains were inhibited, showing inhibition zones ranging between (18-33 mm). Therefore, 600 ppm of Dichloroxylenol is the MIC that inhibits all the tested fungal isolates.

Table 5 Identification of other fungal species, isolated from archeological marble.

No. Fusarium oxysporium Rhizopus oryzae Cladosporium herbarum Alternaria alternate Stachybotrys chartarum

1 1 1 2 ND ND

2 1 ND 1 ND 1

3 ND 1 3 3 1

4 ND ND ND ND ND

Total 2 2 6 3 2

ND: no data recorded.

Table 6 Identification of bacterial and actinomycetes isolate obtained from archeological marble.

No. B. brevis Strept. thermophilus B. coagualns Nocardia

1 1 1 1 4

2 ND ND ND ND

3 1 ND 1 ND

4 ND ND ND ND

Total 2 1 2 4

ND: no data recorded.

Table 7 Minimum inhibitory concentration (MIC) of five microcides for inhibiting all fungal species isolated from infected marble.

Fungal strain Mean diameter of inhibition zone (mm) of fungal species

Dichloro-xylenol Thymol Penta-chlorophenol Sodim Azide p-cresol

(600 ppm) (700 ppm) (400 ppm) (100 ppm) (600 ppm)

Aspergillus niger 25 19 30 24 21

Aspergillus flavus 26 25 31 23 20

Aspergillus fumigates 24 21 31 20 20

Aspergillus sulphureus 22 20 35 21 19

Aspergillus oryzae 18 22 35 20 24

Aspergillus humicola 21 22 30 21 21

Aspergillus clavatus 26 19 28 22 25

Aspergillus parasiticus 33 20 32 25 20

Penicillium stolenferme 22 21 28 19 19

Penicillium oxalicum 25 22 34 20 20

Acremonium murarum 21 19 31 23 20

Acremonium kilinese 19 21 32 20 21

Acremonium fusidiodes 18 19 30 18 22

Fusarium oxysporium 30 35 19 31 20

Rhizopus oryzae 24 21 19 24 21

Cladosporium herbarum 25 20 20 20 19

Alternaria alternata 25 20 22 22 21

Stachybotrys chartarum 32 26 19 18 21

Results of Thymol microcide showed that at concentrations up to 300 ppm, no inhibition was detected. At 400 ppm, only Fusarium oxysporium was inhibited, while at 600 ppm five fungal species were inhibited. Therefore, 700 ppm was found to be the MIC that could inhibit all the tested fungal species.

Results of penta-chlorophenol showed that most fungal isolates were inhibited at concentrations of 100 and 200 ppm, while 400 ppm inhibited all tested fungi, with a mean diameter of the inhibition zone between 19 and 35 mm. Therefore, it can be concluded that 400 ppm represents the MIC of penta-chlorophenol that inhibits all the tested fungal strains.

Sodium azide at concentrations up to 50 ppm had no effect on all isolated fungi, except for Fusarium oxysporium. At 100 ppm, all isolated fungi were inhibited where the mean

diameter of inhibition zone ranged between 18 and 31 mm. Therefore, it can be concluded that 100 ppm was the MIC of sodium azide to inhibit all the tested fungi.

Data of p-cresol showed that no inhibition was detected at concentrations up to 500 ppm, with an exception to Asp. oryzae and Asp. clavatus. At 600 ppm all fungal isolates were inhibited where the mean diameter of the inhibition zone ranged between 19 and 25 mm. Therefore, 600 ppm of p-cresol was the MIC that inhibits all the tested fungal species.

Since each of the tested microcides had a varying effect on each individual fungal strain, it was necessary to indicate the ideal MIC for each fungal strain, individually. These data are shown in (Table 8). For example, 50 ppm of penta-chlorophenol is sufficient to inhibit only Asp. sulphureus, but

Table 8 Ideal minimum inhibitory concentration (ppm) of five microcides for each fungal species isolated from infected archeological marble and resulting inhibition zone diameter (mm).

Fungal strains Di-chloroxylenol Thyamol Penta-chlorophenol Sodium azide p-cresol

ppm Dim2. PPm Dim. ppm Dim. PPm Dim. ppm Dim

Asp. niger б00 25 lOO 19 1OO 19 100 24 б00 21

Asp. flavus soo 19 б00 1S 1OO 2O 100 2З б00 20

Asp. fumigatus б00 24 lOO 21 1OO 21 100 20 б00 20

Asp. sulphureus 500 19 lOO 2O 5O 2O 100 21 б00 19

Asp. oryzae б00 1S lOO 2O 1OO 22 100 20 500 1S

Asp. humicola б00 21 lOO 22 1OO 21 100 21 б00 21

Asp. clavatus 500 2З lOO 19 1OO 19 100 22 500 19

Asp. parasiticus 2OO 1S lOO 2O 1OO 2O 100 25 б00 20

Pen. stolenferme 500 2O б00 21 1OO 1S 100 20 б00 20

Pen. oxalicum б00 22 б00 19 1OO 22 100 20 б00 19

Acr. murarum б00 19 lOO 2O 1OO 2O 100 2З б00 20

Acr. kilinese б00 19 lOO 21 1OO 21 100 20 б00 21

Acr. fusidiodes 500 2O lOO 19 1OO 19 100 1S б00 22

Fus. oxysporium 4OO 19 4OO 21 4OO 19 50 22 б00 20

Rhizopus oryzae 5OO 2O lOO 21 4OO 19 100 21 б00 21

Clado. herbarum 5OO 2O lOO 2O 4OO 2O 100 20 б00 19

Alternaria alternate 5OO 2O lOO 2O 4OO 22 100 22 б00 21

Stachybotrys chartarum 4OO 2З б00 19 4OO 19 100 1S б00 21

Dim.: Diameter of inhibition zone in mm.

Table 9 Ideal minimum inhibitory concentrations (MIC) of five microcides for inhibiting all bacterial species isolated from infected marble.

Bacterial strain Mean diameter (mm) of inhibition zone

Di-chloro xylenol Thymol Penta-chloro phenol Sodium Azide p-cresol

500 ppm б00 ppm 200 ppm 100 ppm 500 ppm

Bacillus brevis 21 2З 2S 2З 19

St. thermopiles 19 21 20 20 1S

B. coagulans 20 19 21 19 19

Nocardia 22 21 19 20 21

asteroieds

Bacteria and actinomycetes isolates were grown on nutrient and starch nitrate media, respectively.

to inhibit the other fungi, it is necessary to use 400 ppm of the same substance.

Against isolated bacteria and actinomycetes

Table 9 summarizes the results of using five microcides with gradual concentrations on isolated bacterial strains from the infected marble.

Dichloroxylenol had no effect at concentrations up to 400 ppm on any of the isolated bacteria, and only 500 ppm was found to inhibit all isolated bacteria and actinomycetes where the mean diameter of inhibition zone ranged between 19 and 22 mm.

Similarly, thymol had no effect on the isolated bacteria and actinomycete up to 500 ppm, and only 600 ppm was sufficient to inhibit all the tested bacterial and actinomycetes isolates where the mean diameter of inhibition zone ranged between 19 and 23 mm. Therefore, it can be recommended to use Thymol at 600 ppm as the MIC to inhibit all the tested bacterial and actinomycetes.

Table 10 Effect of some compounds on removal of microbial spots on infected marble surface.

Tested substance Results of decolorization

Amm. carbonate + EDTA +

1, 2 Dioxan —

Diethyl ether —

Acetone (95%) —

Ethyle alcohol (95%) —

H2O2 (6%) +

Microbial lipolytic enzyme ++

For penta-chlorophenol, 100 ppm inhibited only Bacillus brevis, while 200 ppm was the minimum concentration to inhibit all the tested bacteria and actinomycetes with a mean diameter of the inhibition zone ranging between 19 and 28 mm.

Sodium azide inhibited only Bacillus brevis at 50 ppm, and all isolated bacterial and actinomycete strains were inhibited at

100 ppm with a mean diameter of the inhibition zone ranging between 19 and 23 mm.

Finally, for p-cresol could inhibit all of the four isolated bacteria and actinomycete at 500 ppm, producing inhibition zones with diameters ranging between 18 and 21 mm.

Similar results were reported by Sahaba and Sohair (1988) who found that benlate and thymole microcides were effective to prevent the germination of Aspergillus candidus and Tricho-derma viride on paper even at a concentration of 25 ppm. Leznicka, (1992) demonstrated that p-hydroxybenzoic acid ethyl ester (PHB, Aseptine A), in combination with silicone resins, were effective to control the biodeterioration of monument stones caused by fungal and algal infections.

From the above results, it can be concluded that 100 ppm of sodium azide was the best treatment to stop the growth of all microbial isolates using the lowest possible concentration. Therefore, it was used in the following experiment as described below.

Treatment of the infected marble sample

Results of treating the infected marble piece with sodium azide (100 ppm) showed that it was efficient to completely stop the growth of all microbial isolates, which was shown from the results of swabs taken after 48 h and 6 months giving no micro-bial growth. Colored spots, however, were still observed even after microbial growth was terminated. Result of de-coloriza-tion step showed that microbial enzyme preparation was the most effective to remove the color, followed by H2O2 (6%) and ammonium carbonate + EDTA solution, while the rest of the chemical did not have any effect on the color spots of the marble piece (Table 10 and Fig. 1). Similar results were reported by Konkol et al., (2009) who used the enzyme laccase from the fungus Trametes versicolor and showed to be potential as a decolorizing agent. This study suggests that enzymatic decolorization may be applicable to stains on culturally significant marble caused by microbial colonization.

In addition, Cleere (1997) used a solution of hydrogen peroxide H2O2 to clean brown stains on Carrara marble statue. Result showed no survival microorganisms were detected after treatment.

References

Ammar, M.S., El-Deeb, A.A., 1992. A suggested new formula for the treatment of microbial biodeterioration occurring inside Tut Ankh Amon Tomb and similar antiquities objects. Egyptian Journal of Microbiology 28 (1), 28-91.

Brantner, A., Peiffer, K.P., Grein, E., 1993. Antibacterial assays of the pharmacopoeias: diffusion tests of neutral substances and evaluation. Journal of Planta Medica 59 (7), 675.

Cappitelli, F., Nosanchuk, J.D., Casadevall, A., Toniolo, L., Brusetti, L., Florio, S., Principi, P., Borin, S., Sorlini, C., 2007. Synthetic consolidants attacked by melanin-producing fungi. Applied and Environmental Microbiology 73 (1), 271-277.

Chang, T.T., Li, Ch. Y., 1998. Weathering of lime stone, marble and calcium phosphate by Ectomycorrhizal fungi and associated microorganisms. Div. forest protection. Taiwan Journal of Forest Science 13 (2), 85-90.

Claudio, M., Franco, B., Sara, B., Lorenzo, B., Fabrizio, C., Fabrizio, I., Mauro, C., 2009. Deterioration of medieval painting in the chapel of the Holy Nail, Siena (Italy) partially treated with Paraloid B72 14th International Biodeterioration and Biodegradation Symposium 63 (7), 844-850.

Cleere, C., 1997. L'Innocenza Perduta (Lost Innocence): conserving a Carrara Marble Statue. Journal of Conservation and Museum Studies. 2, 31-34.

Costa, E.W.B., Watson, A.J., Hindson, W.R., 1956. Preservation for rot proofing paper. Australian Journal of Applied Science 7, 113-118.

Cowan, S.T., Steels, K.J., 1974. Manual for the Identification of Medical Bacteria, secon ed. Cambridge Univ. Press, 166.

Danin, A., Caneva, G., 1990. Deterioration of limestone walls in Jerusalem and marble monuments in Rome caused by Cyano-bacteria and Cyanophilous lichens. International Biodeterioration 26 (6), 397-417.

Domsch, K.H., Gams, W., Anderson, T.H., 1980a. In: Compendium of Soil Fungi, vol. 1. Academic Press, pp. 21-328.

Domsch, K.H., Gams, W., Anderson, T.H., 1980b. In: Compendium of Soil Fungi, vol. 2. Academic Press, pp. 541-747.

Doronina, N.V., Lee, T.D., Ivanova, E.G., Trotsenko, Y.A., 2005. Methylophaga murata sp nov.: a haloalkaliphilic aerobic methy-lotroph from deteriorating marble. Russian Academy of Science and Microbiology 74 (4), 440-447.

El-Mougith, A.A., 1999. Effect of benomyl on the growth and lipid composition of Trichoderma koningii. Folia Micorobiologica 44 (1), 41-44.

Gilman, J.C. (1974). A manual of Soil Fungi. Indian Edition published by arrangement with the original American publishers Iowa State University press, USA, pp. 217-251.

Gorbushina, A., Krumbein, W.E., Panina, L., Soukharjevsky, S., Wollenzien, U., 1993. Role of black fungi in colour change and biodeterioration of antique marbles. Geomicrobiology Journal 11, 205-221.

Holt, J.G., 1986. Gram-positive bacteria other than actinomycetes. In: Bergey's Manual of Systematic Bacteriology, 2. Williams and Wilkins, Baltimore, MD.

Konkol, N., Mcnamara, Ch., Sembrat, J., Rabinowitz, M., Mitchell, R., 2009. Enzymatic decolorization of bacterial pigments from culturally significant marble. Journal of Cultural Heritage 10 (3), 362-366.

Lamenti, G., Tiano, P., Tomaselli, L., 2000. Biodeterioration of ornamental marble statues in the Boboli Gardens (Florence, Italy). Journal of Applied Psychology 12 (3-5), 427-433.

Leznicka, S., 1992. Antimicrobial protection of stone monuments with p-hydroxybenzoic acid esters and silicone resin. Laboratorio Nacional de Engenharia Civil, Lisbon, Portugal, pp. 481-490.

Macedo, M.F., Miller, A.Z., Dionisio, A., Saiz-Jimenez, C., 2009. Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin. Microbiology 155 (11), 3476-3490.

Monte, M., 2003. Experimental evidence of oxalate formation by fungal strains on marble samples. Molecular biology and Cultural Heritage. Taylor & Francis, pp. 263-266.

Sahaba, Sohair, Y., 1988. Physiological studies on microorganisms isolated from deteriorated old manuscripts. M.Sc. thesis. Dept. Agric. Microbiol, Fac. Agric., Ain Shames University, Cairo, Egypt, pp. 107-112. Stanley, T., Bryant, M.P., Pfernning, N., Holt, J.G., 1989. Actinomy-cetes. In: Bergey's Manual of Systematic Bacteriology, vol. 4. Williams and Wilkins, Baltimore, USA, pp. 2144-2147. Sze, P., 1998. Biology of the Algae. third ed. Georgetown University, pp. 24-54.

Urzi, C., Lisi, S., Criseo, G., Pernice, A., 1991. Adhesion to and degradation of marble by a Micrococcus strain isolated from it. Geomicrobiology Journal 9 (2-3), 81-90. Winkler, E.M., 2002. Stone in architecture, properties and durability. In: 3rd Completely Rev. and Extended Edition. University ofNotre dame, pp. 14-25.