Introduction

During the last two decades, churches have been an important topic for microbial community studies because of their architectural values and with special focusing on their paintings (Rölleke et al. 1996; Piñar et al. 2009; Capodicasa et al. 2010). Walls, paintings, and even water in the churches were exploited for the identification of the microbial communities inhabiting these niches (Jurado et al. 2002; Radaelli et al. 2004; Portillo et al. 2008; Sterflinger and Pinzari 2012). Most of the microbial deterioration studies have focused on frescoes, mural paintings, and stone monuments (Ciferri 1999; Gaylarde et al. 2011; Imperi et al. 2007; Piñar et al. 2001; Portillo et al. 2009). However, on account of their value, just a few studies describing the biodeterioration agents of paintings on wood panel or canvas have been done, and most of them were control paintings artificially populated with different types of microorganisms (Capodicasa et al. 2010).

A specific characteristic in orthodox churches and rites is “kissing of the icons,” a gesture with religious significance that has been mainly focusing on century-old paintings that are being though as having miraculous powers. Kissing and touching icons (paintings on wood or glass) are an important source of additional bacteria. This extra source of microorganisms can contribute to degradation and deterioration of paintings. Furthermore, this practice causes hygienic problems and can contribute to the spread of some pathogenic bacteria from one person to another, taking into account that many sick people visit such places.

The wooden church from Nicula monastery complex (Fig. 1) dates back to the seventeenth century and is declared a national historical monument under the CJ-II-m-A-07722 code. The church was made from oak and was moved to Nicula in 1973 from its initial place at Gostila. It was exposed for more than three centuries to wind, temperature variations, and more importantly, to humidity, especially its external parts. The interior icons are made out of wood and date from the nineteenth century. They do not present a high rate of degradation and, apparently, are in good preservation state.

Fig. 1
figure 1

The wooden church from Nicula (Romania)

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The aim of this study was the identification of microbial communities inhabiting the Nicula wood church (Cluj County, Romania) and its icons. The investigation also included the associated fungi to bacterial community.

Material and methods

Art work description and sampling

In orthodox churches, the narthex (vestibule) is in direct contact with the external environment. Therefore, to avoid external contamination, our samples were taken from the nave (central part of the church). Nave of the church is separated from the narthex by a wall, and from sanctuary, it is separated by an iconostasis. Lateral walls of the nave have a small window.

Uncultivable microorganism

One sample of degraded wood from a lateral wall (not closed to the window) was taken from the church with the purpose of characterizing the uncultured bacteria.

Cultivable microorganism

On the walls of the church nave, icons are painted on glass and on wood. In the current study, five icons on wood painting were analyzed. Our study included five wooden icons from the nave: two placed on the entrance wall of the nave and three on the iconostasis. Icons were not very close to the door or windows. In order to investigate the bacteria from icons, due to interdiction of sampling wood from these artifacts, the icon surfaces were brushed with rinsed swabs. Two swabs from each icon were stored at 4 °C and plated after 12 h on luria broth (LB) solid media. The plates were incubated at 25 °C.

Cultivable fungi

Six sterile swabs were brushed on the church walls, and one different swab was brushed on each icon. The swabs were used to inoculate Sabouraud media (40 g/L dextrose, 10 g/L peptone, 20 g/L agar, pH 5.6) and incubated for 7 days at 25 °C.

Molecular characterization of the microbial community

DNA extraction and PCR amplification of 16S rDNA from uncultured sample

DNA from a wood sample (0.4 g) was extracted with the Bacteria DNA Preparation Kit (Jena Bioscience) according to manufacturer’s recommendations. The sample was crushed before DNA extraction. The PCR mixture (35 μL) contained 1× Green GoTaq® Flexi Buffer (Promega), 0.2 mmol/L each deoxyribonucleoside triphosphate, 2 mmol/L MgCl2, 1 μmol/L of each primer 27F- AGA GTT TGA TCM TGG CTC AG (Lane 1991) and 1407R- GAC GGG CGG TGW GTR CA (Lane et al. 1988), and 1.25 U of GoTaq® DNA Polymerase (Promega). The PCR amplification was performed at 95 °C for 5 min, 35 cycles, with each cycle consisting in 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s, and a final extension at 72 °C for 10 min. A negative control without DNA template was amplified in the same conditions. The PCR mixes were loaded into an agarose gel (1 %) in TAE buffer (40 mmol/L Tris–acetate, 1 mmol/L EDTA, pH 8.0) with ethidium bromide (0.5 μg/mL). PCR products (~1,400 bp) were excised from the agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega).

16S rDNA cloning

The 16S rDNA pool amplified by PCR from uncultured sample was cloned using the CloneJET™ PCR Cloning Kit (Thermo Scientific) according to manufacturer’s recommendations. The ligation mixture was introduced by transformation into Escherichia coli cells strain XL1 blue (Chung and Miller 1993). About 96 colonies were randomly picked and cultured in 5 mL of LB media for plasmid DNA extraction. The plasmid DNA was sequenced with vector sequencing primers (pJET1.2F and pJET1.2R).

Molecular identification of the cultured bacteria

In order to analyze the cultivable bacteria, colony PCR was performed under the same conditions described for the uncultured sample. The PCR product was purified from an agarose gel. Sequencing was done via a custom sequencing service (Macrogen Company, Amsterdam). The PCR products from cultured bacteria were sequenced with the 27F primer.

Identification of fungi

Genomic DNA was extracted from isolated colonies using the Animal and Fungi DNA Preparation Kit (Jena, Bioscience) according to manufacturer’s instructions. PCR reactions were done with universal fungal primers ITS3 and ITS4 (White et al. 1990). PCR products (330 bp) were excised from the agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The DNA fragments were sequenced using the same primers used for PCR amplification.

Sequencing and sequence analysis

The 16S rDNA gene sequences were manually checked using the free analyzing software BioEdit version 7.0.9 (Tom Hall, Ibis Biosciences). The sequences were compared with the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) using BLASTN (basic local alignment search tool) to retrieve similar sequences.

Results

Uncultivable microbial community

A total DNA extraction method was performed for a wood sample from the Nicula church. From the 16S rDNA library, 96 clones were sequenced. Analysis of the data obtained for these samples revealed the existence of Bacillus species (Table 1). Among the sequences, the predominant species were Bacillus thuringiensis (47 %) and Solibacillus silvestris (20 %). About 15 % of the clones represented less that 98 % identity when compare with sequences from NCBI nucleotide databases. At genus level, they are close to the Bacillus and Solibacillus genera. One percent of the clones showed 98 % similarity with Paenibacillus amylolyticus and 5 % with Paenisporosarcina quisquiliarum.

Table 1 Bacteria obtained from uncultivated sample
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Cultivable bacteria

The cultivable bacteria from five icons were identified using colony PCR using 16S rDNA. A total of 66 colonies were analyzed (colonies were chosen by colony morphology). From these analyzed colonies, a total of 35 unique strains were identified using partial 16S rDNA sequence analysis (Table 2). The analysis of cultivated bacteria indicated also the predominance of Firmicutes, Bacillus genus. A number of 11 unique sequences belonged to Actinobacteria and 8 to Proteobacteria. The most common species found using both approaches (cultivated and uncultivated) are Brevibacterium frigoritolerans and P. quisquiliarum.

Table 2 The diversity of cultivated bacteria
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Fungi associated with bacterial community

Fungal isolates were recovered from walls and icons. Based on morphology, about 38 colonies were selected for identification. From these samples, seven different fungi genera were identified on wooden walls and icon surfaces (Table 3). The Alternaria, Penicillium, and Aspergillus genera were identified on wall and icon surfaces. The Cladosporium, Coprinellus, and Chaetomium genera were identified only on the walls. The Rhodotorula slooffiae was identified only on icons.

Table 3 The diversity of fungi
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Discussions

From uncultivated sample, the sequence analysis of the 16S rDNA library from a wood sample showed the prevalence of the B. thuringiensis strain IAM 12077, S. silvestris strain HR3-23 and P. quisquiliarum strain SK 55. From cultivated bacteria, the presence of Planomicrobium and Variovorax genera was not previously reported in paintings or on wood; the strains are most probably of environmental origin. The fungal genera Alternaria, Penicillium, and Aspergillus were found on church walls and icons.

The Bacillus is a diverse genus, including hundreds of strains (Porwal et al. 2009; Capodicasa et al. 2010). This predominance can be explained by its prevalence in environmental samples and its ability to survive in harsh conditions (Fajardo-Cavazos and Nicholson 2006; Osman et al. 2008) by forming endospores. Another explanation of the prevalence of Bacillus in wood could be the presence in its genome of genes encoding cellulose degrading enzymes or laccase-like enzymes (Khalid et al. 2012). B. thuringiensis is Gram-positive spore-forming bacteria that can produce cellulases, which can mediate the breakdown of cellulose (Lin 2012). Sphingomonas suberifaciens is a soil bacterium that can cause a corky root disease in lettuce, indicating that it may have some lignin-degrading enzymes (Krishnamurthi et al. 2009). P. amylolyticus identified in the sample is known for its capacity to degrade biomasses, especially those containing pectin, due to the pectate lyase enzymes (Boland et al. 2010). The presence and the predominance of Bacillus strains were revealed by both the culture-independent and culture-dependent approaches. Most of the isolated bacteria are of environmental origin. Among these species are bacteria known to be present on wall paintings, such as Arthrobacter, Bacillus, Paenibacillus, Staphylococcus, Pseudomonas, Micrococcus (Pepe et al. 2010), Sphingomonas (Seves et al. 1996), and Curtobacterium of rock paintings (Urzì et al. 2010). Paenibacillus xylanyliticus is known as a xylan-degrading bacterium that can use xylan as a unique source of carbon (Rivas et al. 2005). Bacillus genus was also found present on paintings (Capodicasa et al. 2010; Pepe et al. 2010), mural paintings (Heyrman 2003), and frescoes (Radaelli et al. 2004).

Human skin microbiota is very abundant in Proteobacteria species and Firmicutes represented by the Staphylococcaceae family (Grice and Segre 2011). The presence of Staphylococcus cohnii, Staphylococcus hominis, and Pantoea on icons is of human microbiome origin. These species can have high methicillin resistance and resistance to other antimicrobials, posing problems in interpersonal exchanges and also in the transfer of antibiotic resistance genes to other species (Garza-González et al. 2011), but are not involved in wood degradation. The Micrococcus is very abundant in air, as many as two thirds of the airborne bacteria being micrococci (Kooken et al. 2012), and contributes to the interpersonal exchange of different strains. The genera Streptomyces, Paenibacillus, and Bacillus were found in bacterial communities on wooden art objects and air microflora and have revealed cellulolytic activity (Pangallo et al. 2007). In a more recent study establishing the characteristics of microbial communities isolated from indoor artworks, the same author showed the presence of predominance of Bacillus and Staphylococcus genera with biodegradation properties (Pangallo et al. 2009).

Fungi are considered the most important microorganisms contributing to the biodegradation of the wood samples due to their cellulolytic and proteolytic activities. The presence of fungi is important not only for wood degradation but also for painting degradation. The presence of Alternaria, Aspergillus, Penicillium, and Cladosporium genera identified in the present study are always predominant in indoor and outdoor atmospheres (Rivas et al. 2005). According to Ciferri (1999), the bacteria of the Bacillus genus cannot grow on paintings by themselves, but fungi as Aspergillus and Penicillium promote the further survival of bacteria on paintings. It has been proposed that fungi are first colonizers of the paintings, and the wood and their metabolic products are used by bacteria (Capodicasa et al. 2010). Fungi like Alternaria, Aspergillus, and Cladosporium genera are highly abundant in air. It is estimated that emission rate per person-hour in indoor airborne is 7.3 × 106 fungi genome copies for total particle mass (Qian et al. 2012).

To the best of our knowledge, this is the first study which did a microbiological evaluation of the wooden churches from this part of Europe. Our study will contribute to a better understanding of the new and old microbial community involved in wood biodegradation and to establishing preservation strategies of wood monuments.