Molecular Diagnostics and Determining of Biodeterioration Risk for the 16th Century Icon “Descent into Hell” from the State Tretyakov Gallery

Abstract

Various heritage objects can be subjected to various types of biodegradation and biodeterioration. Mold fungi can destroy many types of art—be it monumental art or easel paintings. Tempera paintings on wood are at risk of biodeterioration, since the wide variety of organic and inorganic materials in art objects often provide an optimal habitat for biological colonization, causing aesthetic and structural damage. In this regard, timely identification and characterization of their microbiological destructive potential are critical. The fungi Syncephalastrum sp. STG-160 and Cladosporium sphaerospermum STG-161, isolated from bio-lesion sites of the 16th century icon “Descent into Hell” from State Tretyakov Gallery, Moscow, were identified and characterized morphologically and molecularly in our work. Syncephalastrum sp. was found in an unusual habitat that has not been previously described for this species. To determine the biodegradability of the identified fungi, their cells were inoculated onto mock layers—egg yolk ochre, cobalt green tempera pigments, and watercolor black. The results show that some pigments were more degradable than others. The addition of cobalt green completely inhibited STG-161 growth and significantly deceleratedSTG-160 mycelium development, most likely due to the presence of heavy metal ions in the pigment. Ochre, a frequently used pigment in restoration practice, is the most degradable material for Syncephalastrum sp. STG-160. Combining culture-dependent methods with SEM and fluorescence microscopy allowed us to identify an invisible individual spore of Syncephalastrum sp. STG-160 and a single hypha of Cladosporium sphaerospermum STG-161 directly on the icon’s surface in clean-contaminated zones, potentially allowing their development in cases of adverse temperature and humidity conditions. Therefore, in order to ensure rapid and effective conservation, it is crucial to assess and quantify the presence of biological systems causing damage to the heritage object itself as well as its individual art components.

1. Introduction

Tempera paintings on ancient wooden icons suffer a series of evolutionary deteriorations and degradations over time due to improper storage and use conditions. This can correspond to the physical state of the structural–functional elements, degradation of the chemical nature of the art components, or mold-induced biodeterioration, causing aesthetic and structural deformation [1,2,3]. Wood and canvas, serving as the base for easel painting, are prepared by craftsmen according to traditional canons. There is a mixture of inorganic materials (pigments, gypsum, and chalk) in organic binders (glues, oils, resins, etc.). Glues can be of animal (gelatin, albumin, casein, and wax) and plant origin (starch, resins, gums, and gluten) [4,5]. These components are rich sources of nutrients for the development of harmful microorganisms (like bacteria, fungi, algae, and lichens) [6,7]. It must also be taken into account that the colonization and proliferation of microorganisms on heritage materials are influenced by microclimatic conditions such as relative humidity, temperature, and light [8]. The characterization of microbial communities through the detection and identification of microorganisms present on cultural assets can be carried out by means of specific complementary methods, and new processes and strategies are constantly being developed [9]. A combination of culture-dependent and culture-independent methods such as next-generation sequencing (NGS) and physico-technical analysis (SEM, FTIR, and Raman spectroscopy, etc.) allow us to estimate the contribution of individual microorganisms to the biodeterioration of an artwork’s pigments, coating layers, and protein binders.

Among the many harmful microorganisms, fungi are especially dangerous due to the fact that their hyphae can proliferate inside heritage materials, spreading several meters in some species, and their dormant spores are often present and can germinate under appropriate temperature and humidity conditions. Furthermore, fungi can produce carboxylic acids (e.g., oxalic, citric, malic, acetic, gluconic, and tartaric acids), which can enhance biochemical degradation [9,10]. A wide variety of fungi are abundant in deteriorated objects, causing darkening, depigmentation, cracks in the paint layer and gesso, and other unaesthetic manifestations. These include fungi of the genera Alternaria, Aureobasidium, Chaetomium, Cladosporium, Curvularia, Drechslera, Fusarium, Gliomastix, Mucor, Penicillium, and Trichoderma [11,12], as well as xerophilic fungi, such as Aspergillus halophilicus, A. domesticus, A. magnivesiculatus, and A. vitricola, which are able to grow at low water activity [13], and even rare representatives of the class Pezizomycetes (Iodophanus sp. STG-150) [14]. The suitable substrate for biodeterioration is the primary substrate, be it a parchment manuscript, a painting, or a monument. In order to study the deteriorative activity of specific microorganisms on a certain art material, research is conducted on mock layers. Since, in most cases, it is impossible to create an exact copy of the material due to the passage of time, mock layers with individual comprehensive analogues of the materials are created, and microbial isolates are inoculated onto it.

There are numerous works devoted to the study of biodeterioration of individual pigments, primers, binders, and supports [15,16]. One such study investigated the biodeterioration and chromatic alterations of painted and gilded mummy cartonnage at the Saqqara Museum storeroom in Egypt. Microbial colonization tests were performed on aged cartonnage replica samples made from materials of similar composition to ancient pigments found in the cartonnage [17]. After the sampling of microorganisms from the object and culturing them, the individual mock layers with pigments, a ground layer, gum arabic as the binding medium, and the linen textile support were contaminated separately by cartonnage-biodegrading fungi and bacteria, which allowed a conclusion about the contribution of these microorganisms to individual materials to be drawn. Another multidisciplinary study of bio-deteriorated Celje Ceiling, a tempera painting on canvas in an old Counts’s mansion in Slovenia, included an investigation of paintings heavily infested with mold in wooden frames [15]. It was found that the presence of a coating layer, protein binders, and pigments, such as goethite, ultramarine, and kaolinite, had an important role in mold development. Therefore, in order to find the right strategies for scientific restoration, it is important to correctly assess the presence of destructive microorganisms on a painting and to determine the spectrum of their “food” preferences regarding pigments, binders, and varnishes.

2. Materials and Methods

2.1. The Object of the Study

The object of our study is the 16th century icon “Descent into Hell” (inv. 19884), 78 cm × 64 cm × 3 cm. This icon was received by the State Tretyakov Gallery (STG) from the Cathedral Old Believer Church of the Dormition of the Most Holy Theotokos in Moscow (Figure 1) in 1936. This cathedral was one of the first to be built in Moscow after Tsar Nicholas II issued a Decree strengthening the principles of religious tolerance in 1905, and it was intended to become a symbol of the revival of the Old Believers in Rus’. The five-domed cathedral was striking in the grandeur and monumentality of its architectural forms. The bell tower had twelve bells, the largest of which weighed approximately 6 tons. The building underwent several changes over the time: the First World War, the October Revolution in 1917, and the final closure and repressions of the church’s rectors during Stalin’s time in 1930.

The icon shield visually consists of two boards, fastened with transverse dowels; the technique is tempera and gilding (Figure 2) in accordance with the Gallery inventory books with records dated by the year 1936. The icon had a darkened top layer of the protective coating, as well as numerous cracks and chips on the paint layer. In the left part of the icon was a large area with graying of the pigment layer and the formation of whitish inclusions of unknown genesis. The STG Restoration Council decided to conduct a molecular diagnosis of the surface and wooden ends of the icon to confirm or refute the biological nature of the damaged areas.

2.2. Isolation and Growth of Fungi Strains

In March of 2024 ten microbiological samples in problem and control areas were collected from the front side and from the wooden ends of the icon (Figure 2). Further manipulations with samples were carried out as described [14]. Then, aliquots were inoculated onto 3 types of slanted agar media: (i) potato dextrose agar (PDA, g/L: potato extract (200 г)—4, glucose—20, agar—20, pH 5.6); (ii) lysogeny broth (LBA, g/L: bactotriptone–10, bacterial yeast extract—5, NaCl—10, agar—20) and (iii) yeast extract peptone dextrose (YPD, g/L: bacterial yeast extract—10, peptone—20, glucose—20, agar—20).

2.3. Phenotypic Characterization of Fungi Strains

2.3.1. Light and Fluorescence Microscopy

The morphology of conidiospores and hyphae in PDA medium was studied using the Carl Zeiss Jena microscope (Jena, Germany) at magnification ×1000. The detection of the fluorescent signal of the stain Calcofluor White (CFW, Sigma-Aldrich, St. Louis, MO, USA) was achieved through Celena X microscope (Logos Biosystems, Anyang-si, Korea), equipped with a specific filter set comprising an excitation filter 375/28, an emission filter 460/50, a lens plan apo fluor oil coverslip corrected 100×, with a numerical aperture of 1.25 and a working distance of 0.19.

2.3.2. Scanning Electron Microscopy

Microstructural characteristics of cultivated filamentous fungi and micro scrapings from the icon’s surface paint layer in areas suspected to biodeterioration and in clean-contaminated zones were examined by scanning electron microscopy (SEM) using a Tescan Amber FIB-SEM ion-beam scanning electron microscope (Tescan Group, Brno-Kohoutovice, Czech Republic). For this purpose, representative samples were chosen, which were further mounted on an aluminum objective table using conductive carbon tape. Then, the samples were placed in the vacuum chamber of the microscope, and the air was evacuated from it until the working pressure reached about 1.0–1.5 × 10⁻⁵ mbar. An Everhart-Thornley secondary electron detector with a focal length of about 5 mm was used to examine material surface. To minimize the impact of the electron beam on the samples structure, their surface was scanned at a sufficiently low accelerating voltage (1 kV). Due to the relatively low electrical conductivity of the samples under investigation, the magnification, as a rule, was limited to the range of 250–15,000 times. To intensify charge flow, a carbon film was deposited on the analyzed material surface with a compact coating Desk Carbon Coater system (NanoStructured Coatings Co., Tehran, Iran).

2.4. Genomic DNA Extraction

The genomic DNA (gDNA) was extracted from lyophilized fungal pure cultures by phenol–chloroform method [18]. Lyophilized samples were resuspended in 200 μL of TES buffer (100 mM Tris-HCl, 1% SDS, 1 mM EDTA, pH 8.5) with glass beads (D = 500 μm) and incubated at 65 °C for 1 h. After cooling, 200 μL of phenol (saturated with 0.2 M Tris-HCl, pH 8.5) was added. Afterwards the upper layer of the sample containing gDNA was washed with an equal volume of the chloroform/isoamyl alcohol mixture (24:1) and then precipitated by 96% chilled ethyl alcohol after 3M potassium acetate (pH 5.2) addition (1/10 of the total volume). All procedures were conducted on ice with periodic shaking (Vortex mixer, Glassco, Ambala, India). The target gDNA precipitate was washed with chilled 70% ethyl alcohol and dried out.

2.5. PCR Amplification

PCR amplification was performed using primer pairs for fungi isolates STG-160 (PV645139.1) and STG-161 (PV636606.1) to hypervariable regions of rDNA (i) Internal Transcribed Spacer (ITS)–ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [19]; (ii) for locus Calmodulin–CMD5 (5′-CCGAGTACAAGGARGCCTTC-3′) and CMD6 (5′-CCGATRGAGGTCATRACGTGG-3’) [20] and (iii) for locus Actin–ACT-512 (5’-ATGTGCAAGGCCGGTTTCGC-3’) and ACT-783 (5’-TACGAGTCCTTCTGGCCCAT-3’) [21].

2.6. Sequencing

The subsequent extraction of amplified fragments PCR was conducted utilizing the CleanupMini kit (Evrogen, Moscow, Russia), in accordance with the manufacturer’s guidelines. Subsequently, the PCR-fragments were subjected to sequencing using the Sanger method, employing the Big Dye Terminator v.3.1 reagent kit (Applied Biosystems, Inc., Waltham, MA, USA) on ABI PRISM 3730 genetic analyzer (Applied Biosystems, Inc., USA), in accordance with the manufacturer’s instructions. The primary analysis of the obtained sequencing spectrograms was performed using Chromas software, version 2.6.6 (https://technelysium.com.au/wp/chromas/ (accessed on 8 February 2024). The nucleotide sequence was subjected to further analysis using Vector NTI v.6.0 [22,23]; the sequence fragment containing a representative reading zone was compared in BLAST+ 2.17.0 (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 21 July 2025) against available sequences in databases. Following a thorough comparison of the results, it was determined that the sequence under investigation is likely to be part of the rDNA of a specific organism.

2.7. Mock Layer Design and Inoculation

Mock layers were developed with modern analogs of art materials used in tempera paintings in the 16th century, according to the literature sources [24,25]. The structure of art layers was the same as described earlier: The wooden support was covered with canvas and glued with a ground layer (water solution of chalk and sturgeon glue, 70 g/L); afterwards, various art materials were applied on the top [11]. Fungal isolates STG-160 and STG-161 were inoculated onto three paint layers: two tempera pigments in egg yolk (ochre and cobalt green) and one watercolor black layer, composed of madder (an organic dyestuff derived from the roots of the Rubiaceae family) [26], burnt Prussian blue (iron(III) hexacyanoferrate(II) [27]), and soot as carbon black [28], with gum arabic as the plasticizer. Each mock layer was approximately 2 cm × 2 cm. The mock layers were inoculated as previously described [11]. Before inoculation, the fragmented pieces were placed into sterile Petri dishes and saturated with 0.3 mL of H₂O per 1 cm³ at 25 °C for 48 h. The pieces with mock layers were placed on sterile hydrophobic spacers such that the lid of the Petri dish did not touch the surface of the fragment. Then, the samples were inoculated with the fungal suspension (suspended in 0.9% NaCl) to an optical density of OD₆₀₀ = 0.35.

Fungal growth on mock layers was measured on the 2nd, 4th, 6th, 8th, 10th, and 12th days. The dynamics of the biodeterioration of mock layers was estimated as the relative average growth area (AGA), as determined by the following formula:

AGA % = [S_tav/S_c] × 100

(1)

Here, S_tav indicates the average area of the STG-160 or STG-161 colony (mm²); S_c indicates the surface area of the whole mock layer (mm²). Measurements were performed in triplicate and repeated at least twice. The results are presented in the form of a thermal scale.

3. Results

3.1. Phylogenetic Identification and Morphological Characterization of Fungal Strains STG-160 and STG-161

The sample cultivation of isolates nos. 1, 2, 7, and 10 did not yield any microorganism growth. Isolate nos. 3, 4, 8, and 9 exhibited bacterial growth. Isolate no. 5 exhibited bacterial and fungal growth of the order Capnodiales (class Dotideomycetes) (Figure 2). Isolate no. 6 only exhibited fungal growth of the order Mucorales (class Mucoromycetes) (Figure 2). Phylogenetic analyses of the ITS region and actin locus of fungal isolate no. 5 and no. 6 exhibited 100% identity with respect to Cladosporium sphaerospermum STG-161 and Syncephalastrum sp. STG-160 (GenBank accession numbers PV636606.1 and PV645139.1, respectively).

Cladosporium sphaerospermum STG-161 was found to be a dark olive green color with a dense consistency when cultivated on a PDA medium (Figure 3a,b). Moreover, light and fluorescence microscopy revealed classical morphological forms of Cladosporium, in addition to conidiophores, such as ovate conidia and ramo-conidia (Figure 3c,d), as previously reported in [11,29]. SEM images show a dense biofilm of Cladosporium sphaerospermum mycelium (Figure 3e,f).

Syncephalastrum sp. STG-160, incubated for seven days at 26 °C on PDA medium, formed a circular colony (Figure 4a) that was gray and simultaneously transparent, with filamentous growth bearing distinct spherical heads (sporangia) (Figure 4b). The dark gray area is a result of the increasing age of the colony and the associated production of spore carriers; the same phenomenon was reported in [30,31]. Transmitted-light and fluorescent microscopy clearly show different morphological forms—including sporangia carriers and vesicles that are covered by merosporangium with spherical merospores (Figure 4c,d). SEM images reveal Syncephalastrum sp. mycelium with conidiophores and conidia heads bearing young merosporangia (Figure 4e,f).

3.2. Mock Layers Biodeterioration

In order to determine the biodeterioration degree, Cladosporium sphaerospermum STG-161 and Syncephalastrum sp. STG-160 were inoculated onto mock layers with modern analogs of art materials used in 16th-century tempera paintings. Tempera pigments in egg yolk (ochre and cobalt green) and watercolor black (madder, burnt Prussian blue, and soot) with gum arabic as a plasticizer were selected as the most frequently used materials in restoration practice (Figure 5).

The ochre pigment in egg yolk emulsion is highly bioavailable for Syncephalastrum sp. STG-160, showing 30% layer deterioration on the 2nd day, 75% on the 4th day, and complete coverage by mold on the 6th day (Figure 5b and Figure 6).

Cladosporium sphaerospermum STG-161 is not as active; nevertheless, it still effectively degrades the ochre pigment, and if extrapolated over a longer period, a 2 cm × 2 cm mock layer would be completely consumed in 20 days. Watercolor black pigment is degraded at a slightly slower rate than ochre by STG-160, but more rapidly than ochre by STG-161. Cobalt green pigment significantly inhibits the development of Syncephalastrum sp. STG-160, and it completely prevents the growth of Cladosporium sphaerospermum STG-161.

SEM of micro-scrapings from the area of the Holy Fathers’ faces (Figure 7a) and their chitons (Figure 7c) revealed a single cone-shaped spore of the mold fungus Syncephalastrum sp. STG-160 (Figure 7b) and rare mycelial hyphae of Cladosporium sphaerospermum STG-161 (Figure 7d). Micro-scrapings from other areas of the pigment layer of the icon did not reveal any traces of mycelial mold fungi.

4. Discussion

4.1. Detection of Destructive Fungi in Tempera Paintings

According to the results of microbiological analysis of the isolated samples, the whitish coating zone did not show the presence of fungal degradation. Previous work [32] has shown that the spectral diagnostics of micro-scrapings of the paint layer in this whitish coating zone (Figure S1) most likely corresponds to kaolinite [33,34].

In further experiments, we plan to refine the composition of the materials using analytical methods. Samples taken from the right side of the icon (points 5 and 6) showed the presence of mold fungi after cultivation. The morphology of the single rare filamentous mycelium (Figure 7d) and cone-shaped spore (Figure 7b) on the micro-scrapings in clean-contaminated zones correlate with the images of the cultivated cultures Cladosporium sphaerospermum STG-161 (Figure 3e) and Syncephalastrum sp. STG-160 (Figure 4e), respectively, which allows us to identify these fungi as potentially dangerous. In cases of adverse temperature and humidity conditions, either at the exhibition halls or in the restoration workshop, it is possible that these mold fungi spread over a large area of the painting, which as we know causes cracking and crumbling of the paint layer and perforation of the primer [35,36].

Common types of fungi that are destructive to tempera painting are those of the genera Alternaria, Aspergillus, Cladosporium, and Penicillium [16,17,18]. Therefore, the presence of Cladosporium did not surprise us. However, the soil-born filamentous fungus Syncephalastrum sp. [37] usually attracts the most attention. Usually, Syncephalastrum species, belonging to the class Zygomycetes, present as colonizers and rarely cause human infection [37,38]. When clinically significant, they are usually implicated in cutaneous infections and onychomycosis [39]. However, these fungus genera may cause opportunistic infections in immunocompromised patients [40], which could prove potentially dangerous for restoration art workers if the fungi multiply rapidly.

4.2. Dynamics of Paint Layer Biodeterioration

A common global practice is to conduct restoration work on replica samples made from the art materials of similar composition to ancient pigments [17]. Therefore, information was obtained on the contribution of Cladosporium sphaerospermum STG-161 and Syncephalastrum sp. STG-160 to the destruction of individual paint layers.

Cobalt green showed the lowest degree of biodeterioration compared to ochre and watercolor black (Figure 4 and Figure 5). Cladosporium sphaerospermum STG-161 was not able to feed on this it at all. Syncephalastrum sp. STG-160 only appeared as foci in mycelial germination on the 6th day, but mycelium was morphologically lifeless, as if dried out; its color was light yellow, in contrast to the intensively branched gray mycelium cultivated on the PDA medium. The low growth rate of these fungi is explained by the chemical composition of the pigment. Cobalt green is said to be similar to cobalt blue (CoO·Al₂O₃), except ZnO partially or wholly replaces the aluminium oxide [41]. Moreover, it was observed that the composition of cobalt green varied considerably with 71.5–88% zinc oxide and 11.5–19% cobalt oxide, with fluctuating amounts of phosphoric acid, soda, iron oxide, etc., depending on the manufacturing process followed [41]. In this regard, we attribute the lack of growth or slow growth of mycelium to the general toxicity of heavy metal ions in the paint [42,43].

The mock layer with the black watercolour, which is a mix of water-soluble colored pigments—madder, burnt Prussian blue, and soot in gum arabic as plasticizer—was not massively conducive to Cladosporium sphaerospermum STG-161 colonization (Figure 5a and Figure 6), but was quite preferable for Syncephalastrum sp. STG-160 (Figure 5b and Figure 6). The gum arabic is added to a watercolour to improve its capacity to stick to a surface [17]. It is possible that the identified fungi contain the appropriate enzymes that hydrolyze the polysaccharides that make up gum arabic.

The ochre pigment suffered the most degradation among the tested pigments for Syncephalastrum sp. STG-160, but Cladosporium sphaerospermum STG-161 degraded this pigment at a slower rate. Ochres are variably coloured rocks and soils primarily composed of ferric oxides and hydroxides [41], which are a rather stable and poorly soluble compound. But fungi have adapted to utilize vital iron. A well-known strategy for effective iron uptake is the production and subsequent uptake of siderophores, which are small molecules that act as high-affinity Fe chelators [44]. Another important Fe-uptake mechanism involves a group of specialized membrane proteins. In this high affinity system, the metal is reduced from Fe³⁺ to Fe²⁺ (in order to increase Fe solubility) by membrane-bound ferrireductases, and it is then rapidly internalized by the concerted action of a ferroxidase and a permease, forming a plasma membrane protein complex [45].

The ability to absorb heavy metal ions depends on the set of genes of each individual organism. It was previously shown that the fungus Rhizophagus irregularis, used as a soil inoculant in agriculture, has 30 open reading frames in its genome, which potentially encode heavy metal (Cu, Fe, and Zn) transporters [46]. The current experiment showed that in terms of adaptability to survival, the fungus Syncephalastrum sp. STG-160 has more advantages compared to Cladosporium sphaerospermum STG-161, particularly in terms of ochre and even cobalt green utilization (Figure 6). This illustrates the danger of the spread of even single spores of the identified fungi if the temperature and humidity conditions change.

5. Conclusions

During the molecular diagnostics of the microbiological state of the 16th century icon “Descent into Hell”, different approaches were used: cultivation of fungi isolates, SEM and fluorescence microscopic examination, and phylogenetic identification by ITS region and actin locus. As a result, two taxa of destructive fungi were identified—Syncephalastrum sp. STG-160 (the order Mucorales) and Cladosporium sphaerospermum STG-161 (the order Capnodiales). Mock layer degradation by these fungi was estimated for egg yolk ochre and cobalt green tempera pigments, as well as for watercolor black.

It was shown that some pigments were more degradable than others. Cobalt green significantly delayed the development of Syncephalastrum sp. STG-160, and there was no growth of Cladosporium sphaerospermum STG-161 on the pigment at all. The ochre pigment is most conducive to Syncephalastrum sp. STG-160 growth—the mock layer of 2 cm × 2 cm was completely covered in mold within 6 days. The black watercolor pigment was digested by STG-160 at a slightly slower rate than ochre, but it was digested faster by STG-161 relative to ochre.

A combination of culture-dependent and culture-independent methods allowed us to identify an invisible individual spore of Syncephalastrum sp. STG-160 and single hyphae of Cladosporium sphaerospermum STG-161 directly on the painting’s surface, which can lead to their development in conditions of adverse temperature and humidity.

Further work is planned to include the precise identification of icon’s art materials using analytical methods, production of mock layers with an expanded palette of pigments and binders to determine the risks of their biodeterioration. Since microorganisms can develop resistance to traditional antiseptics over the course of long-term use, such as benzalkonium chloride, research on new biocides is necessary. For this reason, we should aim to test new biocides: nucleoside derivatives, sulphur-containing heterocyclic compounds, and H-phosphinic amino acid analogues. These procedures will make a significant contribution to scientific restoration.