Bio-Restoration of Mural Paintings Using Viable Cells of Pseudomonas stutzeri and Characterization of These Murals

In the 19th-century Egypt had a strong earthquake leads to damage of several mural paintings. Mural paintings in Ali kadkhoda house (El Rabiemaya), in Cairo, Egypt were among the affected. According to these damages the mural paintings were pre-consolidated and covered by medical gauze and animal glue as an adhesive under extremely dangerous conditions. The traditional conservation methodology as hot water, and acrylics that carried on these mural paintings to strip the medical gauze and animal glue showed no positive results and caused removal of the pigments. Viable bacterial cells of Pseudomonas stutzeri, were used with Brothanimal glue media mixed with agar as a delivery system (gel material) to remove the polymerized animal glue only in 3hours at 35°C. The effectiveness of the bio-cleaning test was assessed. The results confirmed the success of this cleaning biotechnology to remove the animal glue as an organic matter without side effects on the mural paintings pigments. The Bio-restoration technique was safe, low-cost, non-invasive, time saving, and risk-free. Silver nano particles were used to sterilization the mural paintings after final step in the bio-restoration process to insure the death of bacterial cells. At the end, the mural paintings were characterized using SEM-EDX, FTIR, and XRD.


Introduction
Monuments and Mural paintings in Egypt, after the earthquake in 1992, were damaged by the strong vibrations, theorize impendence for losing these mural paintings. Figure 1. A committee for protecting the cultural heritage stored all these damage mural with medical gauze and animal glue as an adhesive to prevent any further damage Figure. 2. Some of these mural paintings are left until now without any conservation treatments.
Ali katkhoda house is one of these buildings that its murals were treated by this technique and kept from 1992-2014. This house (known as El Rabiemaya) is located in Darb Al-Hajar Street. The house contains two holes one is larger than the other. All the walls in the second floor are decorated with several mural paintings with different fields. [1].
Several methods were carried out on these murals to strip the medical gauze and animal glue but due to the long period the animal glue had polymerized and any attempts to take it off showed no positive results and caused removal of the pigments as well.
Moreover, organic compounds (such as animal glue and casein) were used as consolidating materials beside the varnish layer applied in 18 th and 19 th century on the painted surface which lead to darkened pigments and bio pigmentation. [2].
The use of bio-restoration process, thus, the only way to solve these two problems and to let the noble mural paintings easy to be characterized. Microorganisms were deemed responsible for the changing in monuments and mural paintings [3][4][5], but on the other hand they have decisive effects in conservation and restoration process [6][7][8][9][10]. Bacteria has cleaning advantages over other treatments methodology such as laser, especially when the deteriorated substances need to be removed are complex [11] never less, Bacteria were able to acclimate themselves to different environmental parameter (pH, temperature, and nutrients).
In this study the bio-restoration technique was proceeded by direct application of viable bacterial cells of P. stutzeri, and agar as a delivery system on the surface of the covered mural paintings. The bacterial cells removed a most of animal glue in only 10hours and the gauze became easy to be removed without causing damage to pigments. After removal of gauze, the mural paintings were characterized by XRD, SEM-EDX, and FTIR.  After final bio cleaning, silver nano particles were used as an antimicrobial agents, Ag nano particle is one of the essential antimicrobial Nano particles, which is used in cultural heritage [17] and for sterilization of mural paintings without causing damage result or colour effect on mural paintings. [18]

Bacterium Cells and Culture Media
Viable cells of Pseudomonas stutzeri ATCC 17589 strain (American Type Culture Collection, Rockville, MD, USA) were used for the cleaning process.

Growth Media
Two different media were used to cultivate Pseudomonas stutzeri cells MI -Broth media (Z699187-Sigma Aldrich) and Broth-animal glue-containing agar media that consist of 0.5g l −1 KH 2 PO 4 , 0.5g l −1 MgSO 4 ·7H 2 O, 4g l −1 animal glue, and 15 g l −1 of agar in distilled H 2 O. (Jeszeová1 et al, 2018). Autoclave used for sterilizing the media at 110°C for 45minutes and incubating in a shaker (300 rev min at 30°C for 48h).

Cultural Methods
Suspensions with about 1x10 10 colony forming unit (CFU) per 1 ml, obtained by inoculating 10 ml of a nightly broth-culture and broth-animal glue culture into 1L of fresh broth medium, and incubating it in a shaker (300 rpm) for 24 h at 30°C. The bacterial cells were washed by phosphate-buffered saline two times, and then were re-suspended in sterile water, pH 6.9; the final cells concentration was above 1x10 10 concentrations cells ml -1 , the cells were used immediately or stored at 4°C during conservation.

Total Adenosine Triphosphate Determination
Total Adenosine triphosphate (ATP) inspect to monitor the growth culture on the scale's laboratory, and the viability of the Pseudomonas stutzeri cells hang during the in situ bio-cleaning processes, were used a specific enzymatic kit (MAK, Acetyl choline esterase, activity assay-kit-sufficient for clorometric tests, Netherlands). A Bio-counter luminometer (1500 P) connected with a (PMT) photomultiplier tube.

Preparing Replica and Artificial Aging
In order to evaluate the effectiveness of the bio-cleaning process, artificial altered specimens, simulating the presence of animal glue were prepared and applied by soft manual brush as a thin layer directly on the upper surface of gauze on replica (size: 25*25*5 cm) of the of mural paintings. Once the animal glue formed a thin film, about 10 cycles of thermal aging cycles were carried out as 8 hours in an oven at 60ºc followed by 8 hours in room temperature. Figure 3. Two aged replica were used in the experiment: the first Characterization of These Murals replica was covered by the Pseudomonas stutzeri which was distributed over the surface and covered by hydrophilic cotton layer and left for different period (30, 60, 90, 120, 180 minutes).
The second application was agar delivery system, Agar was mixed with the media and then applied over the aged replica (0.5 cm high) for (30, 60, 90, 120, 180 minutes).

Bio-cleaning Application on Mural Paintings
Pseudomonas stutzeri cells with broth animal glue were applied on the aged replica with two carriers (cotton, and agar) for different period (60, 120 and180 min) at (35ºC) and 50% relative humidity, using a controlled room chamber. At the end of each treatment, the application supports were removed and the treated areas were carefully washed with a sponge impregnate with sterile distilled water.

Silver Nano Particles
Nano silver particles (30nm) made by (EGNC-Egypt). 0.1g of AgNO 3 adds to 50 ml of distil water and stir for 30 min at 80°C. 0.1M Tri-sodium citrate was added in ultrasonic processors for stationary operation (Hielscher Company, German model UP 200) until the light brown colour appeared.

X-ray Diffraction
XRD Bruker company model D8 used for compounds identification of Mural paintings. It include a reflectometry, features a high-resolution diffraction, in-plane grazing incidence diffraction (IP-GID), in addition to small angle X-ray scattering (SAXS).

FTIR Spectroscopy
Nicolet Magna 75 FTIR spectrometer used for Absorption spectra in the IR region. 32 signal scans obtained on the real fragments of the mural paintings. Only a milligrams of samples mingled in KBr (IR grade, Merck) bead with a diameter of 13 mm approximately.

SEM-EDX
A Jeol (Tokyo, Japan) JSM 5600 LV SEM images were used for examinations. It was equipped with EDX microanalysis detector (an Oxford Instruments 6587). Low vacuum conditions was the perfect conditions for images due to the non-effected changing of the samples.

FTIR Analyses
The analyses by FTIR techniques indicated the presence of substances identifying the animal glue as protein substance, used for covering the mural paintings in the past over the gauze.

Viable Cell Counts and ATP Determination
P. stutzeri growth rate at 30°C on broth containing animal glue and broth medium, it showed the highest cell density (1.6 O. D. 550 and >10.3 log CFU ml -1 ) on the broth -animal glue medium than broth medium (1.23 O. D. 550 and >8.5 log CFU ml -1 ).
ATP content according to relative luminose unit showed that using broth -animal agar medium was higher than broth medium (21 000 pg ml -1 and 18000 pg ml -1 , respectively).
The effectiveness of temperature on the growth of the Pseudomonas stutzeri both on Broth media and broth-animal glue media was assessed by O. D. 560 and CFU for 36h at 10, 15, 20, 25, 30, 35, 40°C.
Cell density increased ascendant by increasing temperatures and was a very marked increase in cell growth at 35°C at 36 h. Figure 5. The effectiveness of pH value on the growth of the Pseudomonas stutzeri both on broth media and broth-animal glue media was assessed by O. D. 560 and CFU at 5.5, 6, 6.5, 7, 7.5, 8 and 8.5.
A significant increase in the cell growth density was observed at pH=7.5. Figure 6.

Application of Bio-restoration Process on the Replica
The two aged replica were used in the experiment (see 2.2) for different periods (30, 60, 90, 120, 180 minutes). As seen in Table 1. Figure 7.

Application of in Situ Bio-restoration Process
The in situ bio-restoration process included the following steps: a. Mechanical cleaning by brushes and wet cotton was applied befor the bio-cleaning process to remove dusts and dirt from the surface. b. Pseudomonas stutzeri viable cells inculated in brothanimal glue agar media were applied over the mural paintings surface by bruch technique layer by layer (0.5 cm high). c. The gel layers were covered by poly ethylen to save the flexibility of agar media and left for 3 hours. d. Portable heating device was used during application to increase the temprature inside house from 28 to 35°C as it was the perfect growth temprature (see 3.2). e. After the application period, the poly ethylen was removed. f. Final step was removing the gel agar and the medical gauze easly without causing damage for the pigments. g. Nano silver particales (30nm) made by (EGNC-Egypt) were applied over the mural paintings surface by spray technique to sterilise the murals. h. Fragments of mural painting pigments were taked for the characterization process. i. Pre-consolidation process by Bolariod B72 (5%) was applied over mural painting pigments before conservation process.

Mural Paintings Structure
Small fragments were taken from the murals paintings from both pigments and ground layers after removing gauze for examination and characterization.

Ground Layer
The XRD analysis showed that the ground layer consists of about 56% Gypsum (CaSO 4 .2H 2 O), 38% Calcite (CaCO 3 ), and traces from Quartz (SiO 2 ) (about 6%) as shown in Figure 9. The SEM-EDX analysis showed that the ground layer consists of two-layers of mortar: a white sheet layer (A) is consist of Gypsum and Calcite with traces of Quartz and Halite, and the layer (B) contains Calcite, Quartz, and Gypsum, while as shown in Figures 10 and 11.

Painted Layer
The SEM-EDX analysis showed that that the main component of the black pigment is Cerussite (PbCO 3 ). The red pigment is a mix of Cinnabar (HgS) and Minium (PbO 4 ). The Yellow pigment is Ochre FeO(OH). nH 2 O mixed with Zinc Oxide (ZnO) and the white pigment is a mix of Cerussite (PbCO 3 ) and Zinc oxide (ZnO), as shown in Figures (12 to15).

Binding Media
The FTIR analysis showed that none of the Functional group in the spectrum match the main groups of the binding media of the oils that usual used in this period, which mean that the pigments were mixed with water and organic media ( Figure 16).

Discussion
Animal glue was used for mural paintings and in conservation process as binders for pigments (Tempera animal glue), or as adhesives. [19].
Several researches have been carried out on the chemical composition of animal glue present on the mural paintings and monuments addition to the exigency of classifying it prior to degradation [20][21][22].
During aging, animal glue on mural paintings deteriorates, which led to create castration, tensions, crumpling, and discoloration. Therefore the removal of the deteriorated glue during restoration without effect on the pigments of mural paintings became a challenge. [23][24][25] Several studies have been discussing the efficiency of a bio-conservation process in archaeological field for removing back crust, degrading organic matter, or consolidating stone and mural paintings [8,15,[26][27]. However, the treatment duration can be changed from a few hours (Roig et al., 2011a, b), to many days, and even, to one month [28,29]. The bio-cleaning by P. stutzeri used to remove organic matters such as animal glue from the protected mural paintings, bacteria applied for different times (2, 3, and 6 hours) GC-MS and PY/GC-MS approved that animal glue and casein removed (85% and 80%, respectively). [9].
Results in this study showed that the growth of P. stutzeri in temperature less than 20°C has no effect and the optimum temperatures for the bacterial growth was between (30 and 35°C). The growth media used in laboratory studies were two different media broth media and broth-animal glue media. The broth animal glue media was high effective for removing animal glue layer.
Additionally, the basis of our results we used two different delivery system cotton and agar. According to the delivery system and the type of application we would suggest to use agar delivery system beyond 180 minutes to permit P. stutzeri to perform most effectively.
In situ, we consider starting with mechanical cleaning to remove dust before using bio-cleaning process. The bio-cleaning process has several advantages; it is a non-destructive technique, used to remove specific substances or altered compounds from the painting, risk free, and has a good performance rate [6,11]. The application started by applying broth agar media inoculated with P. stutzeri all over the mural painting surface with a height of 0.5 cm for 3hour then removing the gel media easily.
Nano silver particles (30nm) were applied by spray technique after removing of medical gauze and animal glue for sterilizing the mural painting against any microorganisms due to using Ag Np. as antimicrobial in several studies. [17,27,28].
After the bio-cleaning presses, the mural paintings have been characterized, the observation by SEM showed that the ground layer consisted of two different layers one of them is the white sheet layer. The internal layer composed of Calcite, Quartz, and Gypsum as a usual structure (Mora, 1974), while the white sheet layer was a mix of Gypsum and Calcite. The EDX analysis of the pigments showed that the black pigment is Cerussite (PbCO 3 ). The red pigment is a mix of Cinnabar (HgS) and Minium (PbO 4 ), the Yellow pigment is Ochre FeO(OH). nH 2 O mixed with Zinc Oxide (ZnO) and the white pigment is a mix of Cerussite (PbCO 3 ) and Zinc oxide (ZnO). All pigments might be applied as a tempera water colours due to the absence of binding media as the FTIR indicated.

Conclusion
A successful Bio-cleaning process by P. stutzeri was used to remove polymerized animal glue that was used previously as an adhesive over mural painting surface. Broth animal glue media was mixed with agar that used as a delivery system to keep a fine smooth gel surface above the mural surface. A various application periods were applied over replica, the best condition for application was180 minutes at 35°C and pH=7.5 was achieved. In suti the viable cells of P. stutzeri was inoculated in the broth media gel agar over the mural paintings layer by layer and covered with poly ethylene, after application period the gel layer was easily removed without any harmful of the mural surface. The cost of the bio-cleaning process viable bacterial cells was lower than the other conventional methods such as enzymes. Furthermore Bio-restoration was highly efficient, safe, non-invasive, risk-free and easy to apply. The mural paintings in Ali katkhoda were unique, the analysis showed that it applied by water colours over white sheet, the pigments were Cinnabar, Cerussite, Zinc oxide, and Ochre as usually used in this period in Egypt.