Bio cleaning Black Crust of culture heritage stone surface in

AbeerFouadElhagrassy / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 3,Sep 2015, ISSN 2349-0780

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Bio cleaning Black Crust of culture heritage stone surface in Mohammed Ali Palace (Manial Palace) by using of Sulfate reducing Bacteria

Desulfovibrio vulgaris AbeerFouadElhagrassy(1)

(1) Lecturer, Conservation Department, Faculty of Archaeology, Fayoum University.

Black crust is a deteriorated surface layer of Culture heritage stone surface, Many methods have been applied to resolve this problem but none of them save the patina noble. In this paper we applied a remediation treatment of sulfate reducing bacteria (SRB), in particular Desulfovibriovulgaris. The D. vulgaris can convert the black crust (Calcium sulfate) into calcium carbonate from which it was originally formed at only 24h treatment, this strain removed 99% of black crust and save the original stone and patina noble. In the same time it consolidate the stone by forming new calcium carbonate, this technology has many advantages its risk free, performance simplicity, adhesion capabilities, cheap and ready to use anywhere any time, and at the end it save the patina noble of the stone.

1. Introduction

Black crust is a deteriorated surface layer of stone material spontaneously formed from the interaction between a calcareous substrate and the polluted atmosphere in a humid environment and in areas sheltered from rainfall (Esbert, et al. 2001, Moropoulou, et al. 1998, Rodriguez-Navarro, et al. 2003). The formation of sulfuric acid on stonework causes the chemical transformation of insoluble calcium carbonate (CaCO3) into calcium sulfate dehydrate or gypsum (CaSO4. 2H2O) (Ausset, et al.,2000, Bugini, et al., 2000, Gauri,1989). During the crystallization of gypsum, airborne pollutants, such as carbonaceous particles, are embedded in the mineral matrix and cause blackening in sheltered areas. None of the available mechanical and chemical treatments devised for the cleaning of stone altered by black crust has proved to be entirely satisfactory (Campanella, 1990). The importance of an efficient and careful cleaning of stone art work for the removal of chemical and biological alterations cannot be overstressed. Indeed, before cleaning, it is fundamental to have knowledge of the type of stone and the characterization of the chemical and biological alterations in order to choose the optimal cleaning inter- vention. The use of solvents and physical methods (like abrasion) for the removal of chemical alterations can affect the sounds tone material and result in low selectivity (Gauri, and Bandyo. 1999). In the past few years, much progress has been made using viable cells able to remove sulphatesfroms tone ornamental surfaces (Cappitelli, et al., 2006, Webster and May, 2006). The key idea of using living cells in the conservation and preservation of works of art is supported by the fact that microorganisms (mainly bacteria) are the most versatile and ubiquitous organisms found on earth, and they appear to be capable of colonizing almost any environment. Even if we know that some microorganisms have a negative effect, many of them are responsible for positive processes such as the degradation of unwanted organic substances (Sorlini, et al. 2010a). Recently various Cultural Heritage stone surfaces have been cleaned of organic and inorganic unwanted materials (Sorlini, et al. 2010b). Biocleaning techniques have been performed on stone (marble, tuff, sandstone, limestone, etc.), on ceramic material (brick-work), on paper materials, and on concrete using specific bioformulations containing Desulfovibrio sp. and Pseudomonas sp. cells. (Ranalli, et al. 2005, De Graef, et al. 2005, De Belie, et al. 2005, Cappitelli, et al. 2006, 2007, Barbabietola, et al. 2012). Until now, positive results have been obtained from experiments conducted on significant historical monuments like the frescoes at CamposantoMonumentale, Pisa, Italy (removal of a cloth firmly glued to the painted layer) (Antonioli, et al. 2005, Ranalli, et al. 2005, Lustrato, et al. 2012), Milan Cathedral facade (removal of black crusts) (Cappitelli, et al. 2006,

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2007), and Matera Cathedral - both in Italy (removal of nitrates) (May, et al. 2008, Alfano, et al., 2011). Other positive results involve the colored lithotypes of Florence Cathedral (removal of black crust) (Gioventù, et al., 2011), the frescoes on the Santos Juanes Church in Valencia, Spain (removal of animal glue residues and salt efflorescence) (Bosch-Bosch- Roig et al. 2013a, 2013b), and original paper specimens from the IstitutoNazionale per la Grafica, Rome (Removal of animal glue) (Barbabietola et al. 2012). In recent decades, an alternative cleaning technology employing sulfate reducing bacteria (SRB), in particular Desulfovibriodesulfuricans, has been proposed by (Atlas, et al. 1988, Gauri and Chowdhury, 1988). These authors reported that D. desulfuricansconverted gypsum to calcite, suggesting that this was both acleaning treatment and a conservation treatment. Recently,this biotechnology has been optimized to avoid sulfide precipitationby employing an improved delivery system and usingthe aerotolerant strain D. vulgaris subsp. vulgaris ATCC 29579(Cappitelli, et al., 2006, Ranalli, et al., 1997). Although in their relevant review (Webster and May2006) claimed that bioremediation of sulfates is very promisingand has received much attention, they also asserted that therisks posed by this technology are still to be addressed and thatits advantages and limitations over other cleaning procedureshave not yet been fully elucidated. (Gauri et al. 1992) used Desulfovibriodesulfuricansfor the cleaning of an old gypsum-encrusted marble statue that was previously consolidated. This was the first time that someone suggested the use of sulfate-reducing bacteria (SRB) for the removal of gypsum. The work of art had to be immersed in a growth medium for 84 h. This method had two drawbacks: firstly, this kind of treatment cannot be applied to large objects, such as buildings, as it necessitates the immersion of the object in a liquid medium; secondly, the consolidation of the statue prior to the treatment becomes mandatory to prevent the statue from undergoing severe damage due to the immersion. Later, (Ranalli, et al. 1997) employed D. desulfuricansand Desulfovibrio vulgaris on two objects, an old marble “horse hoof” and an old marble column. As an improvement over the previous method, the authors used the inorganic material sepiolite as a delivery system. The cells colonized the carrier, allowing a close contact with the surface to be treated and the water that was provided. At the end of treatment, after 36 h, ion-exchange chromatography proved that sulfate removal by non colonized and colonized sepiolite was 20% and approximately 80%, respectively. However, this method still required a long time (10 to 14 days) for the colonization of the sepiolite, and hydrogen sulfide could react with the iron in the medium, forming iron sulfide precipitates. In this study, we successfully used, an integrated biotechnological system that enables the cleaning of stone cultural heritage in open area by using Desulfovibrio vulgaris.

2. Materials and Methods 2.1 The Manial Palace The Manial Palace was built by Prince Mohammed Ali Tewfik (1875—1955), the uncle of King Farouk, between 1899 and 1929. He had it designed in a style integrating European Art Nouveau and Rococo with many traditional Islamic architecture styles including Ottoman, Moorish, Persian, creating inspired combinations in spatial design, architectural and interior decorations, and sumptuous materials. It housed his extensive art, furniture, clothing, silver, objects d'art collections, and medievalmanuscripts dating back to the Middle Ages. The ceramic tile work of the entryway and the mosque were created by the Armenian ceramist David Ohannessian, originally from Kutahya. 2.2 Bacteria, media and culture In this paper the strain D. vulgaris subsp. vulgaris ATCC 29579, an aerotolerant strain that its metabolic processes transforms Sulphate (SO4

-2) into Sulphide (S-2). Strain ATCC 29579 was maintained in the Desulfovibrio DSMZ 63 medium (K2HPO4, 0.5 g liter_1; NH4Cl, 1.0 g liter_1; Na2SO4, 1.0 g liter_1; CaCl2. 2H2O, 0.1 g liter_1; MgSO4 .7H2O, 2.0 g liter_1; DL-sodium lactate, 2.0 g liter_1; yeast extract, 1.0 g liter_1; resazurin, 1.0 mg liter_1; FeSO4 ·7H2O, 0.5 g liter_1; sodium thioglycolate, 0.1 g liter_1; ascorbic acid, 0.1 g liter_1) and incubated at 30°C for 4 days under anaerobic conditions. (Ranalli, et al., 1997) In order to allow easy application of bacteria, to keep a good contact between the cells and the surface to be treated, and to remove the cells after the treatment Bacterial cells are entrapped in Carbogel (CST, Vicenza, Italy) about 10 minutes during gel formation, the cell pellet was suspended in deaerated phosphate buffer supplemented with 0.599 g liter_1 sodium lactate at pH7.0 at a concentration of 108 cells per milliliter. All the manipulations described above were done under anaerobic conditions in a glove box. Application is performed by positioning tissue paper, followed by a coating of the gel with cling paper. The poultice is left on for 24h and then the softened black crust is removed by used a damp cotton swab.

2.3 Stereomicroscope Samples were observed by Wild Makroskop M420 stereo-microscope (Heerbrugg, Switzerland), equipped with an Olympus OM1 camera ( Chicago, USA).




2.4 Fourier Transform Infrared Spectroscopy Fourier transform infrared (FTIR) analyses were carried out by a Nicolet Nexus spectrophotometer (Washington, USA) coupled with a Nicolet Continuum Fourier transform infrared spectroscopy micro-scope equipped with aHgCdTe detector cooled with liquid N2. Spectra were recorded by a Graseby-Specac diamond cell accessory in transmission mode between 4000 and 700 cm-1. 2.5 X- ray diffraction Analysis (XRD) The mineralogical composition of the precipitation calcium carbonate was determined by XRD analysis The purified crystals were examined by X-ray diffraction (XRD powder diagrams) with Philips PW 1140 and Rigaku- MiniflexCa 2005 diffractometers equipped with a Ni Filter and a Cu-Kα radiation source, and identified according to JCPDS and ASTM, 1974, 1981 criteria. The diffraction peak corresponding to planes 104 (d_0.3 nm) was used to determine approximate Mg content of calcite (Goldsmith et al., 1961). 2.6 Scanning Electron microscope (SEM)

The Morphology and Mineralogical composition of the deposited calcium carbonate crystals were investigated using scanning electron microscope. SEM micrographs were obtained using a Jeol JSM 5600LV Model Philips XL 30 attached with EDX Unit, with accelerating voltage 30 K.V. , magnification 10x up to 400.000x and resolution for W. (3.5nm). Samples surface were firstly coated with carbon then with gold.

3 Results 3.1 Stereomicroscope

By using Stereomicroscope the stone of the Manial Palace was characterized as a Dolomitic limestone with curved faces of grains and columnar grain structure as seen in (fig.1).

Fig.1 the view of the sample grains under stereomicroscope

3.2 Fourier Transform Infrared Spectroscopy

FTIR spectra showed a slight degree of surface sulphatation, the sample characterized by the presence of gypsum (3533, 3410, 1622, 1116 and 673 cm-1) and calcite (1798, 1429, 876, and 712 cm-1) addition to dolomite (2981, 2877, 2517, 1798, 876 cm-1), as shown in ( fig.2)

Fig.2 FTIR spectrum of the black crust Layer.




3.3 X- ray diffraction Analysis (XRD)

The data shown that the sample before treatment was consisting of Dolomite CaMg(CO3)2 49%, Calcite CaCO3 39% , Gypsum CaSO4.2H2O 10%, Quartz SiO2 1% as presented in fig. (3-a).After biocleaning treatment the percentage of gypsum was decreased and the data shown only the Dolomite and Calcite, as shown in fig (3-b).

Fig.3 (a) The XRD of the stone sample before biological treatment (b) The XRD spectrum after biocleaning treatment.

3.4 Scanning Electron microscope (SEM)

The SEM-EDS shown that the sample before treatment in consisted of calcite, Dolomite and gypsum, as shown in Fig.4 , after the biocleaning treatment the SEM shown the new calcite existed instead of Gypsum layer as present in fig.5

Fig.4 (a) SEM of the black crust sample showed the Calcite, Dolomite and tabular crystals of gypsum, the EDS spectrum of the black crust sample.

Fig.5 (a-1) the dolomite crystal, (a-2) natural calcite, (a-3) the new formed Calcite crystal after biocleaning treatment, (b) EDS spectrum of the sample after biocleaning.

a b

a b

b a




3.5 Biocleaning treatment

The biocleaning procedure was applied first on a sample taken from the same place, the sample before, during and after the biocleaning is presented in fig.6 (a, b, c) complete removal of black crust was observed after three applications of 12h each, the XRD and SEM- EDS analysis performed on the samples after the treatment confirmed that the gypsum was removed almost completely. As shown before in fig.3 and fig.5.

Fig.6 Biocleaning treatment of the sample (a) the black crust before treatment (b) after treatment for 12h (c) after second application for 12h again (final step).

3.6 Application on Manial palace stone

After the successful result that showed before the D. vulgaris subsp was applied on Manial Palace stone, In order to allow easy application of D. vulgaris subsp it kept in Carbogel applied on tissue paper, followed by a coating of the gel with cling paper. The poultice is left on for 24h and then the softened black crust is removed by used a damp cotton swab. The final result was a very beautiful nobel patina without any risk as seen in fig.7.

Fig. 7 The biocleaning of the Manial Palace stone (a) before treatment (b) after treatment for 24h.

4 Discussion

The analysis data confirmed that black crust of the Manial Palace consisted of Gypsum as was previously reported by (Heselmeyer, et al. 1991, Moropoulou, et al. 1998, Esbert, et al. 2001, Rodriguez-Navarro, et al. 2003). The XRD approved that the gypsum was a major component in the non-treated stone sample also the SEM_EDS showed that the spectrum of the sulfur element was the second higher one after the calcium element, in addition of that the FTIR analysis confirmed that the presence of gypsum was existed. Microscopic observation demonstrated that the stone of Manial palace is a dolomitic limestone.

In the past, a biocleaning treatment by D. vulgriswas applied successfully to remove sulphates from marble (Gauri, et al.1992, Cappitelli, et al. 2005, Cappitelli, et al.2007) and was applied on limestone that is more porous than the marble

a b c

a b




(Polo, et al. 2010, Gomez- Alarcon and Saiz- Jimenez, 2013) but none of them applied his work on real culture heritage stone. In this present study, D. vulgris was applied successfully first on a deteriorated stone sample by using a carbogel as a delivery system for 12 h and applied again for another 12h the result was a clean stone with patina noble.

The XRD of the treated stone showed that the percentage of the gypsum was decreased and the calcite percentage was increased, the Microscopic investigation by SEM showed a formation process of a new calcite. According to (Gauri, K. and Chowdhury, A. 1988) the presence of calcite after Sulfate reducing bacteria cleaning is probably due to microbial calcification activity; calcium ions released by gypsum dissolution react with carbonate from bacterially produced CO2 and form calcite. This step not cleaning stone surface but also consolidate the stone in same time.

According to successful results D. vulgris were applied on the Manial Palace walls by applied on a tissue paper followed by a coating of the gel with cling paper and left for 24h.After application a Nano silver 10nm was applied to insure that there are no more bacteria still alive, and for final sterilization.

5 Conclusion

The Air pollution present a deteriorated phenomenon found in Manial Palace stone, this phenomenon cased the thick black crusts, this work shows that an integrated biotechnological can provide an important solution for black crust. The bioremediation treatment with D. vulgaris successfully removed black crust from limestone and in same time it consolidate the stone by formation a new calcite, this technology has many advantages its risk free, performance simplicity, adhesion capabilities, cheap and ready to use anywhere any time, and at the end it save the patina noble of the stone.

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Biography

AbeerFouadElhagrassy Egyptian Tel: 00201224000071 Fax: 0020846333178

[email protected]: -e Lecture in restoration and conservation of cultural heritage (inorganic materials) specialize in bio restoration fields, Faculty of Archaeology, Restoration Department, Fayoumuniversity, Egypt.


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