1. INTRODUCTION
Textile artifacts, composed of natural organic materials, including cotton, ramie, hemp, wool, and silk, are prone to microbial deterioration depending on conservation conditions. Once deterioration occurs, restoring the original form is challenging (Kim et al., 2011). Microorganisms, particularly fungi, thrive in environments with suitable temperature and humidity, posing a risk to cultural heritage artifacts. Fungal spores and mycelia are constantly present in indoor and outdoor air, with concentrations influenced by environmental factors, HVAC systems, ventilation, hygiene, and human activities (Kasprzyk, 2008; Samson et al., 2010).
Materials such as cotton, linen, and hemp are susceptible to fungal damage, particularly by fungi capable of cellulose degradation. Hydrolysis of cellulose polymers into cellulose into glucose monomers during cellulose degradation can lead to fiber deterioration (Savković et al., 2021). Wool and silk fibers, however, exhibit resistance to fungal attacks owing to specific cross-linking structures, such as disulfide bonds in animal materials (Szostak-Kotowa, 2004). Despite this, fungal species with keratin-dissolving abilities have been reported to deteriorate wool fibers. Genera such as Aspergillus, Chaetomium, Fusarium, Microsporum, Penicillium, Rhizopus, and Trichophyton release enzymes that degrade wool fibers (Agarwal and Puvathingal 1969). Additionally, Kavkler and Demšar (2012) revealed the highest growth of protein-degrading fungi, Cladosporium cladosporioides and Penicillium corylophilum, on wool surfaces, leading to significant effects on wool fiber aging and degradation. Silk, known for its highly stable bonding structure, is a natural fiber resistant to biological deterioration (Szostak-Kotowa, 2004; Otterburn, 1977), however, cracks and gaps may be observed because of the mycelia of Chaetomium globosum (Grbić et al., 2014).
Among previous studies on fungi deteriorating actual textile artifacts in South Korea, Lee et al. (2003) investigated the 16th-century textile artifacts, and Chung et al. (1987) reported on the matrix-degrading enzymes secreted by fungi isolated from fibrous artifacts.
As such, fungal species inhabiting fabrics may damage cultural heritage artifacts as they secrete enzymes that degrade fibers, and hence, it is most important to ensure that the environmental conditions prevent damage caused by microorganisms or pests in the conservation of textile artifacts (Lee et al., 2003). Against this backdrop, this study aimed to investigate the microbial contamination detected on textile artifacts, identify the respective microorganisms and discuss future conservation measures.
2. MATERIALS AND METHODS
2.1. Target objects
Microorganisms contaminating four transmitted textile objects conserved at the Cultural Heritage Conservation Science Center were identified in this study. The A object was a textile object made of silk fabrics in the Joseon Dynasty era, and it is stored in a frame. It was confirmed that a mixture of starch glue and synthetic adhesive was used in subsequent repairs, and samples were collected from the observed area of microbial contamination. The B object was an 18th-century textile object made of silk fibers that was discovered inside a Buddha statue and has been kept at a temple. Samples were collected at each step of fumigation and conservation treatments to be analyzed. The C object was an 18th-century textile object, that has been managed by descendants to this day. Biological damage has occurred on the object and its paulownia box container, and samples were collected from the inner surface of the box. The D object which was also a transmitted object from the Joseon Dynasty era has been conserved and managed by the municipal museum. The D object was composed of timber and lumber materials, and samples were collected from the observed area of biological contamination. Table 1 summarizes the details of each object investigated in this study.
2.2. Sample collection
Using a sterile cotton swab, the contaminant sample in the respective area on the surface of each object was collected. All samples were collected immediately after being brought into the center for conservation treatment. The sample was mixed with 1 mL of sterile distilled water to be left in a shaking incubator for 24 hours to activate the microorganisms. The cotton swab and suspension were applied to potato dextrose agar (PDA, Difco, USA) for culture at 28°C. After approximately three days of culture, a single colony was isolated and further cultured. For the B object sample, the initial concentration of microbial culture was too high; the sample was cultured again after 10 × dilution, then a single colony was isolated.
2.3. Microbial identification
The detected microorganisms were identified by analyzing the sequence of 16 s ribosomal ribonucleic acid (rRNA) regions by using in the case of bacteria cultured in single colonies and the internal transcribed spacer regions by using ITS4 and ITS1 or ITS5 primers and 18 s rRNA (NL1 and NL24 primers) in the case of fungi. ITS followed by comparison using the BlastN Search program of the National Center for Biotechnology Information (NCBI). All processes related to sequencing were conducted by Macrogen Inc. upon commission.
2.4. Fumigation & Cleaning
In the case of B object, microbial investigations were conducted both before and after fumigation disinfection. In a fumigation chamber equipped with a temperature and relative humidity control system (25°C, 53%), a mixed gas of Ethylene Oxide and Tetrafluoroethane, called Hygen-A (E.O.15% + HFC_134a85%), was administered in a sealed fumigation chamber and processed for 72 hr. Additionally, after fumigation disinfection, the entire surface was repeatedly vacuum-cleaned while using an endoscopic camera to remove contaminants, The cleaning results were assessed based on microbial sampling and the level of activation.
3. RESULTS AND DISCUSSION
3.1. Microbial identification result for object A
Fungal contamination was detected on the plastic cover of the surface and fabrics of the A object, and samples were collected in 11 contaminated areas. Penicillium chrysogenum was most frequently detected across most contaminated areas, in addition to the species of the Hansfordia genus and Aspergillus ruber. The Cladosporium genus was also detected in small counts in areas without contamination. On the plastic cover for the embroidery of the A object, five fungal species were identified, including Byssochlamys spectabilis, whose reproduction was observed. A single species of bacterium; Dermacoccus nishinomiyaensis, was also isolated. Table 2 presents the isolated microorganisms.
3.2. Microbial identification result for B object
For the B object with a high level of biological contamination before the fumigation and conservation treatments, samples were collected at eight selected areas. Table 3 presents images of agar streaked with the collected samples, where large amounts of fungi can be seen in all areas of sample collection. Compared to other areas, the areas 1, 3, 7, and 8 displayed higher concentrations of fungi. Additionally, as the microbial concentration was too high to prevent the isolation of single colonies, the samples were 10 × diluted for further culture and subsequent isolation. For each colony, the respective microbial species were identified through sequencing, revealing four fungal species. Among the 4 species, Aspergillus sydowii was detected in all areas of sample collection in large amounts, while Talaromyces palmae, Xylaria acuminatilongissima, and Myxotrichum carminoparum were additionally detected in different areas (Table 5). After disinfection using a fumigation gas, samples were collected from the same areas once more to determine whether microbial contamination ceased or persisted. The results showed that, although the concentration of the detected fungi was remarkably reduced compared to the pre-fumigation level, fungal spores and mycelia attached to the fabric material remained (Table 4). The dominant species in the second sample collection was Aspergillus sydowii, the species with a large distribution in the first sample collection. Hence, fumigation and dry-cleaning were performed once more, and samples were collected from ten areas, with an additional two areas. Across all areas, no microorganism was detected after culture. Table 5 presents the fungi isolated at the first sample collection. After approximately a year, microbial contamination was shown to have reoccurred during storage, and samples were collected from two areas for analysis. Therefore, a single fungal species Penicillium decumbens was detected, which was not among the previously identified species. This was presumed to be due to further contamination of the material upon exposure to a new environment.
3.3. Microbial identification result for C object
The C object, composed of fibrous materials, was contained in a paulownia box, and the same type of biological damage was observed on it as well as the surface of the box. From the collected samples, a total of 31 fungal species were detected after culture, and when visually discernible colonies were cultured once more, five fungal species were identified. The ITS sequencing revealed that all detected species were of the Aspergillus genus, and the list of the isolated species is given in Table 6.
3.4. Microbial identification result for D object
The D objects samples were collected from the surface of the ancient Alice clip. On the inner side of the metallic adornment of the clip, microbial contamination was detected in an area between the patterned plates composed of timber and lumber materials. For the samples collected from five areas, the identified microbial species were shown to belong to the Aspergillus genus, as shown in Table 7.
4. DISCUSSION
The cause of biological contamination found on the objects investigated in this study was mostly of fungal origin, with additional detection of bacteria in the A object. The Aspergillus genus was detected across all samples, particularly high counts in the B object. The species of the Aspergillus genus are fungi known to be commonly distributed in air, and as they are often isolated in the air of the storage space or on the surface of organic artifacts (Hong et al., 2022), there are active ongoing studies. Most species of the Aspergillus genus, including Aspergillus sydowii, the one isolated from the B object, are reported to have secret proteases that can degrade silk fabrics (Rocha et al., 2021). Micheluz et al. (2016) isolated Aspergillus jensenii and other species from the indoor dust and surfaces of books in a library in Italy. The team has published an article on the toxicity of secondary metabolites produced by the isolated species and their impact on biological organisms. Ariyanti et al. (2016) also isolated xerophilic microbial species with a cellulose-degrading ability from paper documents made of mulberry and reported that most of the isolated species were of the Aspergillus genus, in addition to Penicillium and Trichoderma genera. Meanwhile, Lee et al. (2003) conducted wet cleaning of excavated textile artifacts of the Joseon Dynasty era, and isolated the detected fungal species. The highest count of fungi was obtained from cotton fabrics, while the level decreased in silk, ramie, and hemp, in the given order. The isolated species were of the Acremonium, Candida, Trichoderma, Cladosporium, Penicillium, and Fonsecaea genera. The genera Acremonium and Fonsecaea were found across all fabric types, whereas the Candida genus was found solely in silk, a type of animal fiber. This contrasted the result of this study, where the Aspergillus genus was detected across all tested samples, presumably due to differences in conditions such as air quality and seasonal exposure in the respective buried or stored environment. The A object, in particular, had large counts of Penicillium chrysogenum in addition to the Aspergillus genus, while the Cladosporium genus was found even in the areas without contamination. The Aspergillus genus known for its enzyme production, particularly warrants attention in preserving organic artifacts owing to its significance among microorganisms (Hong et al., 2015). The Penicillium genus, on the other hand, is evaluated as causing the most severe damage to artifacts (Kim, 2006). Hong (1992) reported that such fungal species could reduce the tensile strength of silk fibers while increasing the elongation and destroying the morphology of silk fibers. In another investigation on the biological degradation of silk fibers in soil, no fungal species were isolated, but the rate of bacterial reproduction was high, as a result of which fibrous tissues were destroyed with reduced strength (Szostak-Kotowa, 2004), indicating the impact of bacteria as well as fungi on the deterioration of silk fibers. Furthermore, Byssochlamys spectabilis isolated from plastic cover materials showed a teleomorph stage of Paecilomyces variotii with a substantially high rate of reproduction. The species is also known to reproduce well at low water activity (Aw = 0.70∼085) as with the previously mentioned fungal species (Sterflinger and Guadalupe, 2013). The investigated fabrics also demonstrated past uses of glue and adhesive materials, and as the species of the Aspergillus and Penicillium genera are known to be highly capable of degrading starch and gelatin (Lee et al., 2015), they may feed on such materials to cause discoloration and damage on artifacts. Hence, conservation should be conducted in suitable conditions and with thorough disinfection of artifacts. As such, damages to historic artifacts can result from various factors in their buried or stored environments, and the typical approach to prevent such damages involves fumigation. However, the investigation on the B object in this study has revealed that a small amount of active fungal spores may persist on the surface or within the inner areas of artifacts even after disinfection treatments. Furthermore, as samples cannot be collected from every surface area, the possibility remains that active fungal spores may persist beyond those sampled, even if the collected samples do not reveal cultured fungi. For instance, in the case of the A object, the plastic surface cover disrupted air circulation, potentially leading to biological damage from interior humidity or other contaminants despite blocking outdoor air. Hence, alternative methods of conservation or exhibition should be considered. Efforts to prevent such biological deterioration are crucial. The control of environmental conditions is the best means of protecting fabrics (Sagar, 1988). The degree of reproduction of fungi on the surface of artifacts is closely related to the temperature and RH of the environment. Controlling indoor conditions, with a focus on maintaining temperature and humidity, is fundamental (Hong et al., 2015). The diversity of airborne fungi varies with season and time zone (Kim et al., 2019), surface-dwelling fungi may accumulate dust to form microbial communities over time (Hong et al., 2022). Knowledge of the physiology of relevant microorganisms active in biodeterioration is essential for such control (Sagar, 1988). The management of the RH is the most important factor, and it is generally recommended to keep it within 60% for the conservation of internal environments (Seo et al., 2013).
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