Diversity and biodeteriorative potential of bacterial isolates from deteriorated modern combined-technique canvas painting

International Biodeterioration & Biodegradation 97 (2015) 40e50

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Diversity and biodeteriorative potential of bacterial isolates from deteriorated modern combined-technique canvas painting Aleksandar Pavi
c a, Tatjana Ili
c-Tomi
c a, Aleksandar Pa cevski b, Tatjana Nedeljkovi
c c, c a, * Branka Vasiljevi
c a, Ivana Mori
a b c

Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia University of Belgrade, Faculty of Mining and Geology, Ðusina 7, 11000 Belgrade, Serbia Central Institute for Conservation in Belgrade, Terazije 26, 11000 Belgrade, Serbia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 July 2014 Received in revised form 10 November 2014 Accepted 11 November 2014 Available online

Cultivable bacteria colonizing deteriorated modern painting on canvas were identified in order to evaluate their potential to deteriorate organic and inorganic painting's constituents. Different sampling and cultivation strategies enabled isolation of bacteria belonging to nine genera of Firmicutes, Proteobacteria, and Actinobacteria phyla. Overall predominant bacteria were species of Bacillus (51%) and Staphylococcus (36%) genera. Representatives of six different genera (Staphylococcus, Acinetobacter, Agrococcus, Janibacter, Rhodococcus, and Stenotrophomonas) were isolated for the first time from deteriorated canvas. Almost all isolated bacteria produced proteases, esterases, and lipases, which may be involved in deterioration of painting's binders and media. Bacteria expressing endocellulase were reported. Selected bacterial isolates were tested for ability to deteriorate six pigments. All tested isolates were able to grow in the presence of Ivory black, Red and Yellow ochre, as a sole source of phosphate and iron, inducing their fading. The majority of isolates induced solubilization of Zinc white and Cobalt deep green. Cadmium red pigment that inhibited the growth of a half of isolates proven to be the most toxic pigment. Isolated bacteria were equipped with all required metabolic prerequisites in order to pose a threat to the painting as a whole. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Bacteria Bioweathering Cellulase Modern painting Pigment

Introduction Being composed of wide variety of inorganic and organic materials, each art object represents a unique ecological niche which may be subjected to biological colonization that could result in their aesthetic and structural damage. Research on microbial colonization of oil paintings has demonstrated that selective pressure on the structure of microbial communities is largely exerted by a chemical composition of pictorial layer of a painting (Seves et al., 1996; Santos et al., 2009). Materials constituting easel painting, such as cellulose, animal or plant glues, varnishes, or organic binders and media can be utilized as nutrient resources by many microorganisms in order to support growth and metabolic

* Corresponding author. Tel.: þ381 113976034; fax: þ381 113975808. E-mail addresses: [email protected] (A. Pavi
c), [email protected] (T. Ili
c-Tomi
c), [email protected] (A. Pa cevski), tatjana.nedeljkovic@ cik.org.rs (T. Nedeljkovi
c), [email protected] (B. Vasiljevi
c), ivanamoric@ imgge.bg.ac.rs (I. Mori
c). http://dx.doi.org/10.1016/j.ibiod.2014.11.012 0964-8305/© 2014 Elsevier Ltd. All rights reserved.

activities which can lead to fading of pigmented surfaces, weakening and detachment of the painting layers, loss of support ma
nech-Carbo
et al., terial or appearance of stains (Ciferri, 1999; Dome 2009). Dirt, soot, dust, and dead cells deposited over a painting surface, together with environmental contamination by volatile organic compounds emitted through respiration and vapors are also source of nutrients (Ciferri, 1999). Moreover, products generated during bacterial and fungal metabolism, like organic acids and products of depolymerization of painting's components add to nutrient abundance (Capodicasa et al., 2010). Metalcontaining painting pigments can also define the structure of microbial communities dwelling in pictorial surfaces, since a heavy metal present in a pigment might be toxic, and consequently, reduce microbial diversity (Santos et al., 2009). Although the influence of pigments on colonization, survival, and metabolic activities of microorganisms is still generally unknown, almost equally insufficiently are investigated the capacities of microorganisms, especially bacteria, to induce chromatic and chemical alterations of pigments.

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Presence of complex bacterial and fungal communities has previously been reported concerning variety of art objects including cave paintings (Gonzalez et al., 1999; SchabereiterGurtner et al., 2002; Portillo et al., 2008), mural paintings from churches and other monuments (Heyrman and Swings, 2001; Gorbushina et al., 2004; Pepe et al., 2010; Pangallo et al., 2012), and on pictorial surface of antique canvas or wood panel paintings
pez-Miras et al., (Santos et al., 2009; Capodicasa et al., 2010; Lo 2013a,b). Studies on diversity of these communities have usually included the use of metagenomic approaches and presence of multitude of fungal and bacterial genera has been detected. Most frequently isolated bacteria belonged to Bacillus and related genera, while culture-independent approach revealed existence of environment-specific microbial communities (SchabereiterGurtner et al., 2002; Portillo et al., 2008; Santos et al., 2009;
pez-Miras et al., 2013a,b). On the other Capodicasa et al., 2010; Lo hand, studies concerning biodeterioration process of modern and contemporary art objects are still very limited (Cappitelli et al., 2006; Pangallo et al., 2014). Furthermore, biodeteriorative potential of bacterial isolates and their importance in communities dwelling in painting surfaces is scarcely examined. The study of Santos et al. (2009) where it was demonstrated that bacterial populations were in close contact with the surface of the pictorial layer, while fungal populations were located over bacterial biofilm, recognized bacteria as significant biodeteriogen of paintings. Nevertheless, the literature on the interaction of microorganisms, in particular bacteria, and pigments used in making paintings is still limited. During this study, we have had a unique opportunity to perform research on modern canvas painting of internationally awarded and recognized Serbian artist Petar Lubarda, titled “The Battle of Kosovo”, which was suffering from biodeterioration. Employing different cultivation strategies we have identified diverse cultivable microorganisms dwelling in deteriorated painting, and explored metabolic potentials of isolated bacteria to degrade organic and, most importantly, inorganic components of the painting, in order to provide new insights of bacterial role in deterioration of painted surfaces.

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Material and methods Artwork description and sampling Legacy of Petar Lubarda, a pioneer of late modernist abstract painting in Serbian art, was formed after his death (1974), but due to dispute with Lubarda's widow the items of the Legacy were inaccessible for more than 30 years. In 2009 an expert team entered Lubarda's house that was in extremely poor conditions and found, among other items, “The Battle of Kosovo” painting. This canvas painting (dimension 172.5 cm  190.5 cm; dating from 1953; Fig. 1) belongs to the most important phase of painter's creative work. The face and back of the painting were covered by dirt, while paint layer was relatively stable, without craquelures. Filth had mechanically been removed and painting was stored for two years under controlled conditions (relative humidity: 48e60%, temperature: 15.9e25  C) before it was available for sampling and prior to restoration procedure. Existing documentation indicates that artist, well known for his interest in novel painting techniques and technologies, combined painting techniques on “The Battle of Kosovo”. However, on the reverse side of the painting a remark made by Lubarda was found indicating that “special tempera” was used. Five samples (KB1eKB5) were taken from obverse side of the painting from the areas with visible evidence of biological colonization and/or discoloration (Fig. 1) by sterile cotton swabs. To each swab 3 ml of PBS-Tween 20 solution (0.8 g l1 NaCl, 0.02 g l1 KCl, 0.176 g l1 K2HPO4, 0.024 g l1 KH2PO4, 200 ml l1 Tween 20; pH 7.2) were added, and swabs were vortexed. KB1M and KB2M samples were collected by sterile nitrocellulose membrane (HyBond Nþ, Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA; Poletti et al., 1999) from pictorial areas on obverse side in order to validate this sampling method that have not been used on easel paintings. Isolation and identification of bacteria For bacterial isolation three cultivation approaches were used. Firstly, each swab suspension (100 ml) was plated in duplicates onto

Fig. 1. “The Battle of Kosovo” painting and the sampling areas. Sampling areas KB1, KB2, and KB3 (covering approx. 10e15 cm2), located next to the painting's wooden frame, were showing symptoms resembling mold infestation. KB4 and KB5 samples were characterized by the presence of a few brownish spots, 3e4 mm in diameter, and large, irregular shaped stain light-brownish in color, covering approximately 5e6 cm2, respectively. The presence of black stains characterized sample KB1M, while KB2M sample was taken in the area where no clearly visible spots or stains were present. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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ten times diluted Trypticase Soy Agar (1/10 TSA; Becton Dickinson, Sparks, MD, USA) and R2A agar (0.5 g l1 yeast extract, 0.5 g l1 proteose peptone, 0.5 g l1 casamino acids, 0.5 g l1 glucose, 0.5 g l1 soluble starch, 0.3 g l1 sodium pyruvate, 0.3 g l1 K2HPO4, 0.05 g l1 MgSO4$7H2O, 3 g l1 NaCl, 15 g l1 agar, pH 7.0; Reasoner and Geldreich, 1985), and incubated at 30  C. Bacterial growth was followed for 14 days. Additionally, 10 ml of 1/10 Trypticase Soy Broth (TSB) were inoculated with 100 ml of suspensions and shaken at 90 rpm at room temperature up to five days. Enriched cultures diluted to 106 (100 ml) were plated in duplicates onto 1/10 TSA and incubated at 30  C. Colonies with macroscopically different morphologies and appearance were selected for further analysis. Nitrocellulose membranes were transferred onto 1/10 TSA plates under sterile condition and incubated at 30  C. Bacterial growth was followed up to 14 days. All media contained cycloheximide (50 mg ml1) to prevent fungal growth. Genomic DNA was isolated according to Hopwood et al. (1985), and it was used as a template for 16S rRNA gene amplifications, which were performed according to the instruction of manufacturer of Kapa2G Robust HotStart ReadyMix PCR kit (Kapa Biosystems, Woburn, MA, USA). Primers used in PCR reaction were 27f primer (50 -aga gtt tga tcc tgg ctc ag-30 ; Invitrogen, Carlsbad, CA, USA) and 1492r primer (50 -cgg cta cct tgt tac gac tt-30 ; Invitrogen, Carlsbad, CA, USA). PCR product sequencing was performed by BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), following manufacturer's instructions. Using BLAST algorithm (Altschul et al., 1990) GenBank database was searched by obtained sequences, in order to identify isolated bacteria. Sequences were deposited in GenBank under accession numbers HG313890eHG313953. Scanning electron microscopy (SEM) A small sample (approx. 2.5 mm  1.5 mm) from the obverse side of painting was collected with sterile scalpel and stored at room temperature until analysis. Collected sample was loosely attached piece of the painting's pictorial surface and it was the only piece approved to be taken. Sample for SEM was glued to doublesided conductive carbon tab stuck on standard vacuum-clean stub, and it was coated with gold (thickness of 15e20 nm) by sputtering process (Leica EM SCD005 sputtering machine). Sputtering was performed in the vacuum chamber under pressure <0.05 mbar using sputter current of 40 mA, working distance of 50 mm and sputter time of 100 s. Such prepared sample was examined by JEOL JSM-6610LV microscope. An acceleration voltage of 20 kV was used. Functional enzymatic profiling of bacterial isolates Bacterial isolates were tested for production of lipolytic (esterase, lipase, and phospholipase), proteolytic, and cellulolytic enzymes. Production of esterases was assayed in basal medium supplemented with 1% Tween 20 (SigmaeAldrich; Stenheim, Germany) as substrate, while Tween 40 (SigmaeAldrich; Stenheim, Germany) and Tween 80 (SigmaeAldrich; Stenheim, Germany) were used as substrates for lipases (Lanyi, 1988; Bornscheuer, 2002). Plates were incubated for five days at 28  C and the appearance of opaque zone around colonies indicated appropriate enzymatic activity. Lipases production was additionally assayed on Rhodamine B Agar as described by Kouker and Jaeger (1987), with addition of 0.25% Tween 80. Inoculated plates were incubated at 30  C for five days and lipase activity was detected by orange fluorescence under UV light at 350 nm. Egg-Yolk Agar (EYA) was used for detecting phospholipolytic activity as described by Lanyi (1988). EYA plates were incubated for 48 h at 30  C and the

appearance of turbidity zone indicated lecithinase-producing bacterial isolates. Production of proteases was tested on 4% casein bovine milk (CBM) agar and 12% gelatin solid medium (Tindall et al., 2007). The appearance of a clear halo on CBM plates after four days of incubation at 30  C indicated production of proteases. Stab inoculated gelatin medium was incubated for 48 h at 25  C, then placed at 4  C for 30 min, and gelatin hydrolysis was confirmed by fluidity of medium after cooling. Endocellulase production was investigated as described by Cattelan et al. (1999). Briefly, overnight bacterial cultures were inoculated onto M9 minimal solid medium (6 g l1 Na2HPO4, 3 g l1 KH2PO4, 0.5 g l1 NaCl, 1 g l1 NH4Cl, with addition of MgSO4$7H2O and CaCl2 in final concentration of 1 mM and 0.1 mM, respectively, 15 g l1 agar) supplemented with 0.12% yeast extract and 1% carboxymethyl-cellulose (CMC; Serva, Heidelberg, Germany). After five days of incubation at 30  C, plates were flooded by Gram's iodine (Kasana et al., 2008) and the appearance of a clear zone indicated CMC degradation. Bacteria were tested for the ability to grow under nitrogenlimited conditions using Burk's N-free medium (Wilson and Knight, 1952), gelled with agarose (7 g l1). The occurrence of bacterial growth followed for four days at 30  C indicated bacterial potential for oligonitrophilic growth. Bacterial weathering of painting pigments Analysis of pigments used in the painting was unavailable due to strict rules that allow the use of destructive methods only on fragments which cannot undergo conservation. Six pigments selected for bioweathering testing: Zinc white (ZnO; Nevskaya Palitra, Saint-Petersburg, Russia), Cobalt green deep 835 (CoOeZnO; Sennelier, Saint-Brieuc, France), Cadmium red (CdSeCdSe; Ciba, Basel, Switzerland), Ivory black 755 (carbon, calcium phosphate, calcium carbonate; Sennelier, Saint-Brieuc, France), Red ochre 259 (hematite; Sennelier, Saint-Brieuc, France), and Yellow ochre 252 (goethite; Sennelier, Saint-Brieuc, France) were chosen according to literature data related to painter's opus in general, information received from restorers, and results of analyses preformed on other paintings authored by Lubarda from similar or same period of work. Bacterial growth in the presence of each pigment and their ability to induce pigments' discoloration were tested on modified metal toxicity (mMT) agar medium (Kumar Sani et al., 2001) containing 1% glucose in place of lactate, without PIPES (to avoid medium buffering), and with 1 g l1 of tested pigment. Effects of Zinc white and Cobalt green deep on bacterial growth and activity were additionally assayed at higher concentrations (2 g l1 and 3 g l1). TSB-grown bacterial cultures in stationary phase (10 ml) were spotted onto mMT plates, and incubated at 28  C up to five days. Both appearance of zone of clearance around bacterial growth and bacterial colony color change were followed. Furthermore, bacteria were tested for ability to utilize Ivory black, Yellow Ochre, and Red Ochre pigments as sole source of phosphorus and iron. The presence of phosphorus and iron (extracted by HCl) in each pigment was verified spectrophotometrically using phosphomolybdate method (Murphy and Riley, 1962) and phenanthroline assay (Komadel and Stucki, 1988). Bacteria were grown in BushnelleHaas (BH) broth lacking iron and phosphate (0.2 g l1 MgSO4$7H2O, 1 g l1 NH4NO3, 0.02 g l1 CaCl2, pH 7.0), but supplemented with glucose (1 g l1) and a pigment (1 g l1). Bacterial cultures (100 ml) grown overnight in M9 broth, washed twice with sterile water, and diluted to OD600 of 0.9e1 were used to inoculate BH medium. After incubation for five days at 28  C bacteria were pelleted, and supernatants' pH were measured.

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Amounts of phosphates and total iron (Fe2þ and Fe3þ) released from pigments were determined spectrophotometrically (Murphy and Riley, 1962; Komadel and Stucki, 1988). Statistical analysis Metabolic diversity of isolated bacteria was examined by a cluster analysis that grouped isolates into distinctive biodeteriorative groups. Cluster analysis was based on bacterial ability to produce enzymes able to degrade painting's organic components and to grow under oligonitrophilic conditions.

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Frequencies of Bacillus and Staphylococcus isolates able to degrade organic compounds and to grow under oligonitrophilic condition were compared by c2 test (P ¼ 0.05). Risk for biodeterioration of natural pigments (Ivory black, Red ochre, and Yellow ochre) was evaluated according to quantities of released iron and phosphorus during bacterial growth. The differences between Bacillus- and Staphylococcus-related strains in capabilities for pigments biodeterioration were determined according to oneway ANOVA at the threshold level of P ¼ 0.05, and by the Bonferonni test. All analyses were performed using SPSS 20 (SPSS Inc.) software.

Table 1 Identification of bacterial isolates according to partial 16S rRNA gene sequence. The closest taxonomic relatives

Isolates

Accession number of the closest relatives

The similarity (%)

KB1, KB1*, KB2R KB3FRT, KB3.1, KB3.1F

D83372 NR_027521 CP002439 CP002439 HG941670 KJ416929 JQ795891 KJ018991 HG941670 EU071618 KF923963 KJ016246 CP007539

99.2e99.5 99.7 99.7 99.8 99.5e99.9 98.7 96.0 99.4e99.8 99.4e99.8 98.9 99.2e100 99.4 99.5

KF500919 KF730750 DQ286308 JQ183024 KC178715 CP003687 JN592608 JF280125 JF496288 JX035937 EU513393 CP004069 HM055984 NR_042336 NR_042339 KF641852 EU23912 KC113510 KF322125 KJ399985

99.9 99.9 99.7 99.6 100 98.8 99.5 100 100 99.9 99.4 100 99.9 99.8 99.8 99.9 99.4 99.1 99.1 99.2e100

KB1M4D KB1M7 KB2M1 MB2M2 KB2M9 KB2M4, KB2M5

KJ000740 KF032701 GU568201 JQ023599 JN700144 HQ857778 KF500919 KF424264 FJ601909 KC858851 KF032680 GQ141979

99.6 97.7 99.9 99.5 100 100 99.8 98.6 100 98.4 99.7 99.0

Proteobacteria Pseudomonas oryzihabitans S6-242 Acinetobacter lwoffii MTCC 496 Stenotrophomonas maltophilia 262XG5

KB1A*R KB1M5D KB1M1.2

JQ660200 AB859068 KF818624

99.9 100 100

Actinobacteria Rhodococcus qingshengii B2 Janibacter melonis CM2104 Agrococcus jenensis DSM 9580

KB1B*R KB5.1F30 KB2M3ps

KJ028076 NR_025805 AM410679

99.9 99.1 99.9

Firmicutes Staphylococcus schleiferi Cd22-1 schleiferi subsp. coagulans GA-211 pseudintermedius HKU10-03 pseudintermedius HKU10-03 hominis B14 hominis AT4sh hominis M-S-TSA hominis SDT63 hominis B14 hominis EHFS1 epidermidis Y43B/2 epidermidis CC6 aureus NRS 100 Bacillus cereus D7 thuringiensis Y34 thuringiensis serotype H11 thuringiensis SGC cereus w22 thuringiensis MC28 cereus IARI-T-12 cereus MBG23 cereus XA5-11 thuringiensis JDG2 cereus TMPSB-M20 thuringiensis serovar kurstaki HD73 pumilus MB-42 stratosphericus 41KF2a aerophilus 28K cereus B19 cereus KNUC260 thuringiensis EWB-6 cereus NBAII B7 cereus MVK04 thuringiensis DRR-1 cereus TAV2-6 cereus LLCG23 cereus LZ020 cereus L16 anthracis NX28 cereus D7 licheniformis KA5 thuringiensis INRS13 cereus CLHDHF(2)B 1-1 cereus KtRA2-72 Sporosarcina aquimarina SF237

KB1.3T KB1.1R, KB2.4, KB3.1 KB1.2T KB2.2T KB4.1F30, KB4.1R, KB5.1 KB4.2F30 KB1.1F30, KB1.1T, KB1.2F30, KB4.2R KB2.4R KB4.2T KB1.1 KB1.2 KB1.2R KB2TA KB2TB KB2.1F30 KB2.3R KB2.3T KB2.3F30 KB2.5F30 KB3R KB3RF KB3.1F KB3.3F KB4T KB4.3T KB4.4F30, KB4.7F30, KB1M5, KB1M6, KB1M6D, KB2M1.2, KB2M1.3, KB2M6 KB4.6F30 KB5.2F30 KB1M2.1 KB1M3 KB1M4

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Results and discussion Isolation and identification of bacteria Using different sampling and cultivation strategies 64 bacteria differing in colony morphology and appearance had been isolated, and identified by sequencing. GenBank database search revealed that isolates were affiliated to nine genera belonging to three different phyla (Table 1). Taxonomic classification, number of isolates per sampling area, and employed sampling and cultivation methods are given in Table A.1 (Supplementary information). Isolates of the most abundant phylum Firmicutes (90%) shared the closest identity with different species of Bacillus (51%), Staphylococcus (36%), and Sporosarcina (3%) genera. Dominance of Firmicutes phylum, primarily Bacillus-related species, has previously been reported by other authors (Gorbushina et al., 2004; Santos
pezet al., 2009; Capodicasa et al., 2010; Pepe et al., 2010; Lo Miras et al., 2013a,b). However, this is the first report on the presence of non-spore-forming Staphylococcus-related bacteria on damaged canvas painting, which have previously been isolated from the damaged mural painting and frescoes (Gorbushina et al., 2004; Radaelli et al., 2004). The presence of staphylococci on artwork objects is still a matter of scrutiny, as these bacteria are common microflora of the skin and mucosae of humans and animals, and therefore could be considered as a mere contamination. Regardless their likely origin, the fact that they were isolated form the “The Battle of Kosovo” painting stored in a room with limited access, under controlled atmospheric conditions for two year prior it was available for microbial analysis, has suggested that staphylococci might be biodeteriogens to whom an adequate attention has not yet been paid. Phylum Actinobacteria was represented with only three isolates (5%), but they were affiliated to three different genera e Agrococcus, Janibacter, and Rhodococcus. Similarly, three Gram-negative

bacteria of Proteobacteria phylum (5%) were sharing the highest identity with species of Acinetobacter, Pseudomonas, and Stenotrophomonas genera (Table 1). Non-spore-forming bacteria (Staphylococcus, Rhodococcus, Janibacter, Agrococcus, Pseudomonas, Stenotrophomonas, and Acinetobacter) were almost equally frequently isolated as spore-forming (Bacillus and Sporosarcina; Table A.1). In other studies related to diversity of isolated bacteria from the easel paintings majority of bacteria belonged to spore-forming genera such as Bacillus, Sporosarcina, Paucisalibacillus, Virgibacillus, and/or Paenisporosarcina
pez-Miras et al., 2013a,b). (Capodicasa et al., 2010; Lo Sampling by nitrocellulose membrane, which was used for the first time on an easel painting, did not result in isolation of any Staphylococcus-related strain. However, bacteria related to Acinetobacter, Agrococcus, Sporosarcina, and Stenotrophomonas genera, and to Bacillus licheniformis were isolated solely by this approach (Table 1; Table A.1). Moreover, employing membrane sampling method Stenotrophomonas- and Acinetobater-related bacteria, which were frequently detected solely by culture-independent methods on damaged canvas paintings (Capodicasa et al., 2010;
pez-Miras et al., 2013a,b), fresco (Pepe et al., 2011), and manuLo scripts (Michaelsen et al., 2010; Principi et al., 2011) were isolated from such art object for the first time. Using nitrocellulose membrane we also isolated Agrococcus-related bacteria, which have never been detected on canvas paintings, but were isolated from fresco and wall painting (Wieser et al., 1999; Pangallo et al., 2009). For the first time, presence of Janibacter and Rhodococcus genera members on easel painting was confirmed by their isolation. They have never been detected on paintings either by cultivationdependent or cultivation-independent methods, although members of these genera with capabilities relevant for degradation of objects of cultural heritage have earlier been reported (Krakova et al., 2012; Pangallo et al., 2012). Isolation of spore-forming bacteria from all sampled sites (Table A.1) does not necessarily mean

Fig. 2. Scanning electron micrographs performed on sample after coating with gold (high vacuum mode). a) Filamentous morphology and the size suggest that scanned microorganism probably belongs to actinomycetes. White arrow indicates extracellular polymeric substance; b) Coccoid-shaped bacteria; c) Filamentous fungus with conidiogenous apparatus in close contact with pictorial surface of the painting; d) Granulated and pitted pictorial surface covered by fungal reproductive structures and possibly cobweb filaments. Size bar and magnification are indicated at each image.

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that they have existed as metabolically active, living cells, since spores could be deposited onto a painting's surface by different routes, including unintentional transmission by restorer alone. Nevertheless, isolation and cultivation of 28 non-spore-forming bacterial strains strongly indicates that viable microorganisms have been present on pictorial surface of the painting even after two years of storing under controlled conditions which should not favor microbial survival or growth. Scanning electron microscopy analysis SEM analysis of paint layer confirmed the presence of subaerial microorganisms (Fig. 2). The presence of filamentous bacteria (Fig. 2a), and developed and branched fungal structures with spores (Fig. 2c) have implied that these microorganisms were metabolically active in certain period of time. Their partially collapsed and deformed appearance observed at the moment of analysis might indicate a status of standing microorganisms, which could have been active, but could not further develop as environmental conditions have been changed. On the other hand, such

45

appearance of microorganisms could be caused by SEM imaging itself. Observation of an extracellular substance spread over the paint layer (Fig. 2a), similar in appearance to extracellular polymeric substance reported in other studies (Crawford et al., 2010; Diaz-Herraiz et al., 2014), further supports this assumption. In addition, the presence of structures of biological origin, such as cobweb filaments, were detected in close contact with microorganisms (Fig. 2d). The presence of filamentous actinomycetes was evidenced by SEM, but they were not cultivated most likely due to media used in the study, which could not favor growth of this slowgrowing bacteria. Bacterial degradation of organic compounds and growth under nitrogen-limited conditions Although a pictorial surface is a harsh and restricted environment, it is still susceptible to microbial colonization. Only microorganisms with appropriate physiological (production of spores, resistance to xeric environments, etc.) and metabolic capabilities (production of protease, lipase, cellulose, etc.) are able to survive or

Fig. 3. Clustering of bacterial isolates according to functional similarity. Heat map shows grouping of 64 bacterial strains into four main biodeteriogen groups further divided into seven clusters according to their ability to produce enzymes relevant for degradation of painting organic constituents, and their ability to grow under nitrogen-limited conditions. Dark color rectangles indicates presence of activity, whereas lighter color represents its absence. Cas e casein; Gel e gelatin; T20 e Tween 20; T80 e Tween 80; EY e egg yolk; CMC e carboxymethyl-cellulose; NFG e nitrogen-free growth; T40 e Tween 40; Oli e olive oil. Different cultivation strategies: strains cultivated on R2A medium (:), on 1/10 TSA medium ( ), on 1/10 TSA after culture enrichment in 1/10 TSB (C), and isolated using the nitrocellulose membrane palted on 1/10 TSA (-).

▵

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€ hlenhoff et al., 2001; Dar et al., grow under such conditions (Mo 2005). Even though there is evidence that bacterial colonization of the painting surface might occur prior to fungal (Santos et al., 2009), the literature on bacterial involvement in deterioration process is still limited. Therefore, we have tested bacteria for the ability to degrade organic compounds that are used as pigment binders (e.g., oil, egg yolk, methylcellulose) and media (e.g., casein), or painting support (e.g., cellulose), and for the growth under nitrogen-limited condition. The most widespread enzymatic activities among isolated bacteria were esterase, protease, and phospholipase (Fig. 3), which could be responsible for degradation of diverse pigments' binder, media, and varnishes. Finding that bacteria produced more than one lipase emphasized that isolated lipolytic bacteria, presumably Staphylococcus-related isolates, might play significant role in degradation of various painting emulsifiers. In fact, Pangallo et al. (2014) have recently found lypolytic staphylococci and bacilli to be potential bacterial degraders of damaged epoxy resin statue colonized with these microorganisms. Capodicasa et al. (2010) hypothesized that degradation of cellulose canvas fibers was primarily attributed to fungi, since isolated bacteria did not show exocellulolytic activities. But we have tested isolated bacteria for the ability to degrade carboxymethyl-cellulose (CMC) and this study is the first report on isolation of bacteria from the canvas painting with (endo)cellulolytic abilities. Cellulose

degradation abilities were predominantly identified among Bacillus sp. strains which frequently lacked oil-degrading activity. Endocellulolytic activity was also shown for isolates of Janibacter, Pseudomonas, Sporosarcina, Agrococcus, and Stenotrophomonas genera. Surprisingly, even six isolates related to Staphylococcus genera exhibited endocellulolytic activity, which is rarely found staphylococcal ability (Paul et al., 1986). These results, together with earlier finding of intimate interaction between bacterial cells and linen fibers of the canvas (Capodicasa et al., 2010), suggests that bacterial endocellulase may play important role in degradation of cellulose fibers probably together with fungal cellulases. Statistical analysis confirmed observed degradative specialization among isolates belonging to Bacillus and Staphylococcus genera. Although strains of both genera exhibited wide metabolic diversity (Fig. 3), yet significantly higher number of Staphylococcus sp. strains than Bacillus sp. strains showed lipase activity (olive oil) (74% vs. 29%; P ¼ 0.0007, c2 test), while the higher proportion of isolates of Bacillus than Staphylococcus genus was able to degrade CMC (82% vs. 26%; P < 0.0001, c2 test) and to grow under nitrogen-limited conditions (89% vs. 45%; P ¼ 0.0012, c2 test). Enzymatic characterization has demonstrated that most bacteria possess necessary metabolic activities potentially responsible for deterioration of painting's organic compounds, and reveled a great metabolic diversity among isolates belonging to the same genera.

Fig. 4. Changes of painting pigments induced by bacterial isolates. Growth of the bacterial isolates in presence of (a) Ivory black (Acinetobacter sp. KB1M5D), (b) Red ochre (Staphylococcus sp. KB2R), and (c) Yellow ochre (Staphylococcus sp. KB1), without visible pigment fading or colony color change. d) Solubilization of Zinc white pigment (Staphylococcus sp. KB1); e) Solubilization of Cobalt green deep with no colony color change (Staphylococcus sp. KB4.2R); f) Development of colony in absence of Cadmium red pigment (Bacillus sp. KB2M6); Development of (g) red (Bacillus sp. KB2M6), (h) yellow (Bacillus sp. KB2M1.2) or (i) grayish colony (Stenotrophomonas sp. KB1M1.2), when bacteria were grown in presence of Cadmium red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Bioweathering of painting pigments The next step in uncovering the bacterial role(s) in biodeterioration of a pictorial layer was evaluation of bacterial potential to provoke chromatic and structural changes in a painting pigments. Heavy metal-containing painting pigments can define the structure of microbial communities inhabiting a pictorial surfaces, since metals might be toxic, and thus reduce microbial diversity (Santos et al., 2009). Although studies focused on investigation of the influence of pigments on colonization, survival, and metabolic activities of microorganisms are rare and mostly related to the efflorescence phenomenon (Rosado et al., 2013), and to pigments conversion by muralassociated or environmental bacteria (Petushkova and Lyalikova, 1986; Jahn et al., 2006), almost equally insufficiently are investigated the capacities of microorganisms, in particular bacteria, to induce chromatic and chemical alterations of pigments. Relaying on literature data related to Lubarda's opus, and the results of analysis preformed on other artist's paintings we have selected six chemically different pigments (Ivory black, Red ochre, Yellow ochre, Zinc oxide, Cobalt deep green, and Cadmium red) to be analyzed for their susceptibility to deterioration by 38 isolates. Bacterial isolates were selected according to their exhibited biodeteriorative enzymatic profile, taxonomy, incidence, and the area of isolation. Particularly suitable for bacterial growth were pigments Ivory black, Red ochre and Yellow ochre, which sustained the growth of all tested strain in plate assay (Fig. 4aec), without any clear media discoloration or colony color change. These pigments have been used in the art since the Paleolithic Era of human history. Ivory black (or bone black) contains carbon, calcium, and phosphate (Berrie, 2007), while the principal coloring matter in Yellow and Red ochres, is iron(III)-oxide present in form of minerals goethite

47

and hematite, respectively. Being natural earth pigments, they are infrequently pure (Eastaugh et al., 2008), and may contain minerals and/or clay beneficial for microbial growth. Therefore, to further investigate bacterial deterioration of natural pigments we tested, in liquid culture, if natural pigments can be a sole source of essential nutrients, namely phosphate and iron, i.e., whether natural pigments can support bacterial growth and their metabolic activities. Our results showed that all tested bacteria were able to grow in BH minimal broth lacking both phosphate and iron, and to induce pigments' discoloration (Fig. 5a) and media acidification (Table 2). Observed acidification could be a result of release of various organic acids produced during bacterial glucose metabolisms, and at least be partially responsible for phosphorus and iron leaching (Table A.2; Fig. 5bec), and pigments discoloration. The most extreme pH changes were measured for Stenotrophomonas sp. KB1M1.2 (Ivory black, pH 3.62), Staphylococcus sp. KB4.1F30 (Red ochre, pH 2.98), and Bacillus sp. KB2.3T (Yellow ochre, pH 2.35). Statistical analysis revealed that staphylococci released significantly more phosphates and iron from Ivory black (P < 0.0001, the Bonferonni test), and more iron from Red ochre (P ¼ 0.015, Bonferonni test) than bacilli (Fig. 5bec), suggesting that bacteria of Staphylococcus genera could represent higher risk for pigments Ivory black and Red ochre than bacteria of Bacillus genus. Although Bacillus-related bacteria capable of iron leaching from pure mineral hematite have previously been isolated from rock arts (Gonzalez et al., 1999), iron leaching from iron-bearing pigments like ochres and Ivory black induced by different bacterial genera is for the first time reported in this study. In addition, to the best of our knowledge this is the very first study which shows that painting pigments could be exploited as nutrient sources by painting-associated microorganisms.

Fig. 5. Biodeterioration of the pigments induced by bacterial activities. a) Pigments' discoloration during bacterial growth in iron-free minimal medium, without added available phosphates (P) or with available phosphates (þP), and control (available phosphate, without bacteria). Extraction of both phosphorus (b) and iron (c) from different pigments by bacteria related to Staphylococcus (black bars) and Bacillus (grey bars) genera grown in the medium without added phosphorus and iron. Bar presents average values of three measurements. The error bars indicate standard deviations. Statistically significant differences between bacteria of Staphylococcus and Bacillus genera in quantities of released phosphates or iron according to ANOVA (P < 0.05) and the Bonferonni test are denoted.

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48

When grown in the presence of Zinc white or Cobalt deep green (Table 2) the majority of isolates formed colonies, and were able to solubilize both pigments (Fig. 4dee). Out of total number of tested bacteria, 84% and 79% isolates were able to grow on plates in presence of Zinc white and Cobalt green deep, respectively. Almost all of examined bacteria provoked Zinc white (79%) and Cobalt green deep (74%) clearing, indicating pigments' solubilization due to bacterial activities (Table 2; Fig. 4def). The majority of Staphylococcus- and Bacillus-related strains together with Agrococcus sp. KB2M3ps, Pseudomonas sp. KB1*AR, and Acinetobacter sp. KB1M5D isolates, demonstrated growth and solubilizing activities in the presence of both pigments (Table 2). Zinc white is zinc(II)oxide, and it is used as a pigment, lightening or preparative agent for painting

Table 2 The capacities of bacterial isolates to grow in the presence of pigments and to induce their discoloration and medium acidification. ZnWa

CoGa

CdR

pHb Ib

Solid media Firmicutes Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus

Ro

Yo

Liquid media

þ þ þ þ þ þ þ þ þ þ þ / þ

þ þ þ þ þ / þ þ þ þ þ þ þ

/ y y r / / y / / y / / y

4.0 4.2 4.0 5.0 4.0 3.8 4.6 3.9 4.4 3.9 4.1 3.9 4.2

3.7 4.0 4.5 4.5 4.7 4.6 4.9 4.7 4.4 4.6 4.2 3.0 4.2

3.5 3.8 4.4 4.3 4.6 4.2 5.0 4.1 4.4 4.7 4.2 4.4 4.2

þ þ þ þ þ þ þ þ þ þ þ þ / / þ þ þ

þ þ þ þ þ þ þ þ / þ þ / þ þ / / þ

/ y / y y / r / / r r y / y / / /

4.9 4.2 4.8 4.3 4.2 5.3 4.9 5.7 5.9 5.7 5.3 5.8 4.1 4.8 6.0 5.8 5.7

3.7 4.5 4.7 4.7 4.5 4.4 5.1 4.1 4.4 4.5 4.4 5.2 4.3 4.2 4.4 4.3 4.5

2.8 2.4 4.5 3.9 3.9 3.6 5.0 4.3 3.2 3.9 4.2 4.2 4.7 4.4 4.0 3.9 4.1

Sporosarcina sp. KB2M4 Sporosarcina sp. KB2M5

/ /

/ /

y /

4.7 4.0

5.5 4.4

5.0 4.6

Actinobacteria Rhodococcus sp. KB1*BR Janibacter sp. KB5.1F30 Agrococcus sp. KB2M3ps

e / þ

e / þ

r / /

4.6 4.6 5.1

3.8 4.9 4.5

3.4 4.5 4.5

Proteobacteria Pseudomonas sp. KB1*AR Stenotrophomonas sp. KB1M1.2 Acinetobacter sp. KB1M5D

þ e þ

þ e þ

r g /

3.6 3.6 4.2

3.7 4.8 4.0

3.7 4.6 3.9

Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus

sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp.

sp. KB1 sp. KB1.1T sp.KB1.2F30 sp. KB1.3T sp. KB2R sp. KB2.4 sp. KB2.4R sp. KB3FRT sp. KB3.1 sp. KB3.2F sp. KB4.1R sp. KB4.1F30 sp. KB4.2R

KB2TA KB2.3T KB2.5F30 KB3R KB3RF KB3.1F KB3.3F KB4T KB4.3T KB4.7F30 KB5.2F30 KB1M2.1 KB1M7 KB1M4D KB2M1.2 KB2M1.3 KB2M6

ZnW e Zinc white; CoG e Cobalt green deep; CdR e Cadmium red; Ib e Ivory black; Ro e Red ochre; Yo e Yellow ochre; (þ) e appearance of zone of clearance around bacterial colony; () e no zone around bacterial colony; (/) e no growth; y e yellow; r e red; g e grey. a Results of bioweathering tests in solid media supplied with 0.3% pigment; otherwise pigment was 0.1%. b The pH value as a mean value of three replicates of bioweathering tests in liquid media with 0.1% pigment.

support, while Cobalt green deep pigment is made by heating of Co(II)O and Zn(II)O. Pigments solubilization could be induced by release of different organic acids in media, produced during bacterial glucose metabolisms or by acid-metal complex formation (Franz et al., 1991; Madhaiyan et al., 2004). Microbial production of organic acids may present particular risk for pigment degradation, since some pigments are acid- or alkali-sensitive (Gettens and Stout, 1966; Roy, 1993; Ciferri, 1999). In addition, same growing and solubilizing pattern was observed when bacteria were exposed to pure ZnO (0.3%; data not shown), which is well known for its antimicrobial activity against bacteria. These data demonstrated that the most of bacteria tested in this study possess potentials to inflict damages to pictorial layer containing Zinc white and/or Cobalt green pigments. The strongest negative effect on bacteria was observed for Cadmium red (CdSeCdSe) pigment, which inhibited growth of 47% of tested isolates (Table 2). Almost a half of both Bacillus- and Staphylococcus-related strains formed colonies in presence of this pigment, together with Rhodococcus sp. KB1*BR, Pseudomonas sp. KB1*AR, and Stenotrophomonas sp. KB1M1.2. Bacteria that were able to grow in the presence of Cadmium red did not develop clear zone around colony, but changed a colony color into red or yellow (Fig. 4geh). The only exception was Stenotrophomonas sp. KB1M1.2 isolate, where the change of colony color into gray was observed (Fig. 4i). In the study of Pages et al. (2008) it was shown that Stenotrophomonas maltophilia Sm777 strain could reduce selenite to elemental selenium, and accumulating it change the colony color into red. This particular strain was also able to protect itself from toxic Cd forming CdS-cluster in cytoplasm, resulting in yellow colonies. Observation of similar coloration changes among our bacterial isolates might suggest that they were able to accumulate heavy metal(s) and thus tolerate their toxicity. However, Stenotrophomonas sp. KB1M1.2 (Fig. 4i) isolated in this study has exhibited rather different phenotype, changing its coloration into grey. Clearly, detailed studies are required to resolve mechanisms of Cadmium red bioweathering. Out of all tested strains, the ability for growth in the presence of Zinc white, Cobalt green deep and Cadmium red was observed for 45% bacteria, including strains of genera Bacillus (8 isolates), Staphylococcus (6 isolates), Pseudomonas (KB1*AR), Rhodococcus (KB1*BR) and Stenotrophomonas (KB1M1.2). Among them, potential for deterioration of all three pigments was confirmed for six Staphylococcus sp. strains, six Bacillus sp. strains, and Pseudomonas sp. KB1*AR. Conclusions Although it is well known that diversity of a total microbial community cannot be addressed using just one approach, only culture-dependent approach offers a possibility to isolate and directly examine the whole spectrum of biodeteriorative potentials of microorganisms which could inflict a damage or might accelerate further biodeterioration of attacked artwork object. More complete information on metabolic capacities of microorganisms involved in biodeterioration process may give valuable suggestions on the best restorative and preserving strategies, as the saving cultural heritage has become a major concern of many modern societies. Employed sampling and cultivation strategies enabled isolation of bacteria related to nine different genera, of which isolates belonging to Staphylococcus, Acinetobacter, Agrococcus, Janibacter, Rhodococcus, and Stenotrophomonas genera (all non-spore-forming bacteria) were isolated for the first time from easel paintings. Since all of them, except staphylococci, previously have only been detected by molecular methods, this study is the first assessment to

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their deteriorative capacities. Performed metabolic profiling demonstrated that majority of isolated bacteria have necessary capabilities to deteriorate a painting's organic compounds. A several bacteria belonging to seven different genera exhibited endocellulolytic activity, implying their potential involvement in degradation of canvas, probably in synergy with fungi. Enzymatic characterization revealed a great metabolic diversity, even among isolates belonging to the same genera. It was shown that isolated bacteria exhibit different effects on painting pigments. Ivory Black, and Red and Yellow ochres were able to sustain bacterial growth as a sole resource of essential nutrients. Our results confirmed that isolated bacteria possess capacities to deteriorate painting's media and binders, and provided the evidence for their possible involvement in painting's support deterioration as well as in pigments weathering. Acknowledgments This work is a part of research realized within the project No. 173048 financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors are very grateful to Vanja Jovanovic, restorer from CIK, Belgrade for her precious collaboration, constructive advice and suggestions. IM express her deepest gratitude and appreciation to dr Savvetta for finding a time in her busy schedule to get involve in the manuscript writing. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ibiod.2014.11.012. References Altschul, S., Gish, W., Miller, W., Myers, E., Lipman, D., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403e410. Berrie, B.H. (Ed.), 2007. Artists' Pigment. A Handbook of their History and Characteristics. National Gallery of Art, Washington. Bornscheuer, U.T., 2002. Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol. Rev. 26, 73e81. Capodicasa, S., Fedi, S., Porcelli, A.M., Zannoni, D., 2010. The microbial community dwelling on a biodeteriorated 16th century painting. Int. Biodeterior. Biodegrad. 64, 727e733. Cappitelli, F., Principi, P., Sorlini, C., 2006. Biodeterioration of modern materials in contemporary collections: can biotechnology help? Trends Biotechnol. 24, 350e354. Cattelan, A.J., Hartel, P.G., Fuhrmann, J.J., 1999. Screening for plant growthpromoting rhizobacteria to promote early soybean growth. Soil Sci. Soc. Am. J. 6, 1670e1680. Ciferri, O., 1999. Microbial degradation of paintings. Appl. Environ. Microbiol. 65, 879e985. Crawford, R.W., Rosales-Reyes, R., Ramírez-Aguilar, M.L., Chapa-Azuela, O., AlpucheAranda, C., Gunn, J.S., 2010. Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc. Natl. Acad. Sci. U.S.A. 107, 4353e4358. Dar, S.A., Kuenen, J.G., Muyzer, G., 2005. Nested PCR-denaturating gradient gel electrophoresis approach to determine the diversity of sulfate reducing bacteria in complex microbial communities. Appl. Environ. Microbiol. 71, 2325e2330. Diaz-Herraiz, M., Jurado, V., Cuezva, S., Laiz, L., Pallecchi, P., Tiano, P., SanchezMoral, S., Saiz-Jimenez, C., 2014. Deterioration of an Etruscan tomb by bacteria from the order Rhizobiales. Sci. Rep. 4 http://dx.doi.org/10.1038/srep03610.
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