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ISOLATION AND CHARACTERIZATION OF MOULDS DEGRADING MURAL PAINTINGS

Maria Iasmina Moza1, Monica Mironescu2, Cecilia Georgescu2, A. Florea3, Livia Bucşa4

1 FACULTY OF SCIENCES, “LUCIAN BLAGA” UNIVERSITY OF SIBIU, ROMANIA.

2 FACULTY OF AGRICULTURAL SCIENCES, FOOD INDUSTRY AND ENVIRONMENTAL PROTECTION, “LUCIAN BLAGA” UNIVERSITY OF SIBIU

3 FACULTY OF MEDICINE, "IULIU HAŢIEGANU" UNIVERSITY OF MEDICINE AND PHARMACY CLUJ-NAPOCA, ROMANIA.

4 FACULTY OF HYSTORY AND PATRIMONY “LUCIAN BLAGA” UNIVERSITY OF SIBIU, ROMANIA.

Summary The aim of this research was to isolate and characterize the micro fungi that have degraded the fresco (around 75% of the painted surface) in an old orthodox church in the Sibiu area. The main interest was to know which fungi could grow on this substrate under uncontrolled humidity and whether their growth could damage the fresco. The fungal populations attacking mural painting were identified and characterized using microbiological techniques involving cultivation, isolation, optical microscopy (OM) and scanning electron microscopy (SEM). The analysis of fungal colonies and cultures, correlated with the microscopic analysis allowed the identification of moulds as belonging to the genus Penicillium , respectively Penicillium rugulosum and Penicillium crysogenum. Key words: fungal attack, mould, Penicillium, mural painting, biodegradation

iasmina_moza@yahoo.com Introduction

During the recent decades there has been a general concern about the deterioration of historic monuments.The problem of deterioration of works of art is relevant in countries like Romania that are rich in cultural heritage (Boldura, 2007, **). Along with other factors microbial grown plays an important role in this process (Suihko et al., 2007).

Growth of microorganisms on paintings may cause aesthetic and structural damage (Griffin et al., 1991). Frescoes may be colonized by communities of microorganisms that interact with the substrate (Ciferri, 1999). Generally, growth of microorganisms on mural paintings is supported by humidity, slight alkaline pH and the presence of organic and inorganic nutrient sources (Ripka, 2005), compounds as casein from milk are incorporated in the

damp lime plaster (Ciferri, 1999). Microorganisms can induce unsightly discoloration of frescoes, formation of pigmented biofilms, biomineralization and degradation of organic binders leading to structural damage (Herrera et al., 2004).

Saline efflorescence is also promoted by high humidity (Gorbusina et al., 2004, De Souza, 2002) and the presence of bacteria is alarming because it facilitates the emergence of fungi (Mitchell and Ji-Dong, 2000). The specialized literature reports biological attacks produced by microorganisms only in special cases, occurring after an increase in moisture support, as a result of infiltration, flood, excessive touring, etc., or after use of inappropriate materials in restoration (Bucşa et al., 2010), other authors report the activity of microscopic fungi on the

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surface of monuments, which increases their alteration (Aira et al., 2007, De Nuntiis et al., 2004, Hoffland et al., 2004, Berner et al., 1997). Fungal spores sensitive to humidity and heat emit hyphae that penetrate the substrate, with mechanically destructive effects (Berner et al., 1997, Warscheid and Brams, 2000).

Frescoes represent a special case because they are more frequently attacked by actinomycetes than other types of substrate (Gaylarde and Gaylarde 2005). As colonization proceeds, the smooth surface of the painting is modified: pigments become rough and bioreactive (Giullitte, 1995). These events give rise to exfoliation, cracking and loss of the coloured patina (Ciferri, 1999). Their metabolic products cause further chemical damage (Beech, 2004); the capacity of fungi to dissolve carbonates depends on available carbon sources (Hirsch et al., 1995).

Studies based on culture-dependent techniques showed that fungi are predominant species of Penicillium, Aspergillus, and Cladosporium (Guglielminetti et al., 1994). The analysis methods are usually based on the isolation of fungi directly from the frescoes, also aerobiological investigations are applied for the investigation of biological deterioration of cultural heritage (Nugari and Roccardi, 2001).

This research investigates the biodeterioration causes on the mural painting from an old orthodox church in the Sibiu area, in order to isolate the micro fungi present on the fresco and establish which type of fungi could grow on this substrate under uncontrolled humidity and whether their growth could damage the fresco. The fungal populations attacking mural painting were identified and characterized using microbiology techniques involving cultivation, isolation, optical microscopy (OM) and scanning electron microscopy (SEM).

This research is a part of an undergraduate thesis.

Material and methods The old Orthodox Church “Buna

Vestire” from Jina, Sibiu having a mural painting original from 1801 restored in 1987 – 1989 was analysed.

Diagnostic and humidity measuring “In-situ” diagnosis was made, in order to identify the areas with different fungal attack by using visual appreciation (figure 1) (Bucşa, 2009). The relative humidity was also measured, using a contact humidometer, to assess the correlation with this type of moulds attack. Sampling and isolation of microorganism Fungi were isolated from different parts of painted surface and from air of the church using the Koch method. Samples were collected with sterile cotton swabs immersed in sterile water. Czapek–Dox agar and malt agar was used for fungi isolation and characterisation. Isolated moulds growing on the agar plates were transferred by subculturing from hyphal tips, colonies or spores to fresh liquid malt agar for another characterisation. Macro and micromorphological analysis The isolated fungi were identified on the basis of macromorphological and micromorphological characteristics using solid and liquid media, stereomicroscope, OM and SEM analysis, keys for identifications and specialized literature also (Bánhegyi et al., 1985, Dan et al., 1999, Şesan et al., 2007, Tănase et al., 2001, 2006).

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Results and discussion Intensity of attack and humidity values

The mural painting from the entire church is strongly attacked by moulds, the deterioration degree being around 75%

from the painted surface and the humidity value are also very high, more then 70% (table 1) (Bucşa et al., 2010); this values precede the germination of Penicillium spores (***).

The most aggressive attack is observed in narthex and altar. Table 1. Value of the air relative humidity (RH

%) (Bucşa et al., 2010).

Location Hight (m)

Relative humidity (RH %)

Pronaos 2 70 - 75 Narthex 2 75 - 80

Altar 2 82 - 87

Isolation and characterization of microorganism

Eight moulds were isolated from the

church: four from air inside and four from the frescoes samples. Three of them abbreviated after the church walls (JN1, JN2 and JS) were chosen to be investigated because of their frequency on the degraded mural painting. This species have been reported also in airborne fungi. One non-sporulating fungus was also isolated.

The preliminary result of JN1, JN2 and JS identification by traditional morphological methods showed that all three species belong to Penicillium genera. Moreover, fungi of the genus Penicillium were found in samples taken from frescos (De Nuntiis et al., 2004, Aira et al., 2007). Each of the three types of moulds isolated displays a different behaviour on solid and liquid substrate (figure 2) and a short description is shown in table 2 and 3.

A

B

C

Figure 1. A - Narthex, S wall, round fungi colonies, B - Altar bolter, massive fungal

attack, C - Access way, NE wall with saline efflorescence (Bucşa, 2009).

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Table 2. Different behaviour on solid substrate after 7 days.

Media Solid substrate Mould JN1 JN2 JS

Appearance

after 2 days

after 2 days

after 3or 4 days

Colony diameter

70-80mm 60-65mm 50-55mm

Form circular circular irregular Margin complete complete curly Profile umbonate convex flat Colour blue-green green-

blue green

Reverse yellow white white Pigments present absent absent Exudates absent present absents

Table 3 Different behaviour on liquid substrate after 7 days.

Media Liquid substrate Mould JN1 JN2 JS

Appearance

after one day

after one day

after one day

Growing moderate moderate abundant Turbidity moderate dense unimpor-

tant Cellular deposit

absent unimportant unimpor-tant

Colour grey white-grey green-blue

Pigments present present absent Exudates absent present present

Microscopic analysis of isolates species

Using the usual protocol of SEM (Ploaie and Petre, 1979; Flegler, et al., 1993; Goldstein et al., 2003) the figures 3, 4 and 5 demonstrated clear the differences between the three species analyzed. All the three species are terverticilate penicili (figure 3, A, B and C) with short metulae and fialide of different length which release spores.

The comparative analysis of the three species of fungi isolated shows that JN1 fialides have medium-length chains (figure 4, A), as well as JS (figure 4, C) and the arms are much thicker than others two penicili (figure 4, B and C). The spores are round (figure 5, A) as in the case of JN2 (figure 5, B) and not roughs like in case of JS (figure 5, C). JN2 penicili (figure 4 B) are much different as compared to JN1 and JS (figure 4, A and C) by having very long chains fialide; JS coniophore walls have a spiky appearance conferring a better resistance to dehydration (figure 4 and 5, C).

A

B

Figure 2. JN1, JN2 and JS after 14 days of incubation: A - on Czapek-Dox agar, B - on liquid malt

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Figure 3. OM analysis (100 ×): A- JN1- with fuchsine, B - JN2, C - JS

Figure 4. SEM analysis of fialides: A - JN1, B - JN2, C - JS.

Figure 5. SEM analysis of spores: A - JN1, B - JN2, C - JS.

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Conclusion Mural painting from the church

“Buna Vestire” from Jina was investigated. The fungal attack was very high, the deterioration degree being around 75% from the painted surface and the humidity value are also very high especially in the narthex and altar.

Eight moulds were isolate from samples taken from the air inside and painted surface but only three were chosen to be characterized because of their frequency on the degraded mural painting from the church.

The preliminary analysis of fungal colonies and cultures, correlated with the

microscopic analysis allowed to identify the moulds as belonging to the genus Penicillium.

Each of the three types of moulds isolated displays a different behaviour on solid and liquid substrate. On solid substrate JS have a slower growing and his morphological development is different in comparison with the other two. JN2 present pigments and form exudates only on liquid substrate also the turbidity are very dense.

After SEM analysis, JN1 was identified as a Penicillium rugulosum and JS as a Penicillium crysogenum.

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**http://www.arcchip.cz/w08/w08_gomoiu.pdf - Gomoiu Ioana and Mohanu D., State of the Art and Biodeterioration

***http://193.230.235.21/moodle/file.php?file=/41/micotoxicoze_pps/MICOTOXICOZELE.pps