Two new cellulolytic fungal species isolated from a 19th-century art collection

The archive of the Universidad de Costa Rica maintains a nineteenth-century French collection of drawings and lithographs in which the biodeterioration by fungi is rampant. Because of nutritional conditions in which these fungi grew, we suspected that they possessed an ability to degrade cellulose. In this work our goal was to isolate and identify the fungal species responsible for the biodegradation of a nineteenth-century art collection and determine their cellulolytic activity. Fungi were isolated using potato-dextrose-agar (PDA) and water-agar with carboxymethyl cellulose (CMC). The identification of the fungi was assessed through DNA sequencing (nrDNA ITS and α-actin regions) complemented with morphological analyses. Assays for cellulolytic activity were conducted with Gram’s iodine as dye. Nineteen isolates were obtained, of which seventeen were identified through DNA sequencing to species level, belonging mainly to genera Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Penicillium and Trichoderma. For two samples that could not be identified through their ITS and α-actin sequences, a morphological analysis was conducted; they were identified as new species, named Periconia epilithographicola sp. nov. and Coniochaeta cipronana sp. nov. Qualitative tests showed that the fungal collection presents important cellulolytic activity.

Variations in the composition and appearance of a material as a consequence of the action of microorganisms is known as biodeterioration 1 . This phenomenon becomes evident with the presence of reddish-brown or yellowish-brown patches, microfungal structures and textural changes, which are commonly found in ancient documents 2 . These conditions apply to a nineteenth-century French collection of drawings and lithographs by Bernard Romain Julien (1802-1871) that is held in the archive of the School of Plastic Arts of Universidad de Costa Rica. The damage due to the microbial proliferation in these works of art is related to the storage conditions, especially to the damp and warm environments 3 . To design an effective and specific treatment according to the species growing in the laminae led to the isolation and identification of the fungal species responsible for the foxing of the lithographs.
Previous investigations of the microbiota in antique documents reported the presence of fungi that belong mainly to genera Alternaria, Aspergillus, Chaetomium, Cladosporium, Penicillium, and Trichoderma 1,2,4-7 . For instance, El Bergadi et al. (2013) isolated, identified and characterized the microbiota of manuscripts from an ancient collection of the Medina of Fez and found Aspergillus niger, Aspergillus oryzae, Mucor racemus, and Penicillium chrysogenum, as the most frequent species from a total of 31 fungal isolates 8 .
Because of nutritional limitations in which these fungi grew and where cellulose of the laminae was the only source of carbon, the species responsible for the biodeterioration were believed to possess cellulolytic activity 8,9 . This cellulolytic activity is of interest for multiple biotechnological processes, such as treatment of agroindustrial residues 10,11 or production of cellulases 12 . This condition was first deduced and published in 1903 by van Iterson in "La décomposition del la cellulose pas les microrganismes" 7 . An investigation of the microbial diversity in a nineteenth-century Islamic and Koranic book led to the discovery of nine fungal species with the ability to degrade carboxymethylcellulose (CMC), including Aspergillus niger, A. oryzae and Hypocrea lixii 8 . Michaelsen et al. (2009) and Pinzari et al. (2006) described Aspergillus versicolor, A. nidulans, A. terreus and Chaetomium globosum as agents in the microbiological damage of old documents 5,6 .
Given the interest in the developing methods for protecting and preserving ancient documents from microbial degraders 13 and the importance of obtaining microorganisms or enzymes with the capacity to degrade lignocellulosic wastes 14 , the aim of the present work was to isolate and identify the fungal species responsible for the biodegradation of a nineteenth-century art collection and to determine their cellulolytic activity. We found 19 fungal isolates belonging mainly to genera Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Penicillium and Trichoderma. Two samples not identified through their DNA sequences were identified through morphological analysis as new fungal species, namely Periconia epilithographicola sp. nov. and Coniochaeta cipronana sp. nov. Qualitative tests showed that the fungus collection presents important cellulolytic activity.

Methods
Sampling and isolation of cellulolytic fungi. A total of 13 laminae from a nineteenth-century French collection of lithographs belonging to Universidad de Costa Rica with signs of biodeterioration were sampled in areas of critical damage (colored or discolored areas, microfungal structures or other observable textural changes in the paper) with sterile cotton swabs, which were subsequently submerged in Phosphate Buffered Saline (PBS, 100 µL). Samples (50 µL) were cultured onto potato dextrose agar (PDA; Difco Potato Dextrose agar, BD company, France), and onto water agar with carboxymethyl cellulose (CMC, 1%, Sigma-Aldrich) with kanamycin (km, 50 µg/mL, Sigma-Aldrich). Morphologically distinct colonies were isolated and purified onto plates with the same culture media 2,15-17 .
The amplified products were purified with a clean-up kit (EXO-AP, Thermo Scientific, USA) and sequenced with a genetic analyzer (ABI 3130xl) and a reaction kit (Big Dye v.3 Terminator Cycle Sequencing Ready Reaction Kit, Applied Biosystems, USA), using ITS and actin primers (1 μM). Sequences were analyzed with software (MEGA 7), and were run through a Standard Nucleotide BLAST (Genbank, NCBI nucleotide database) to assess the similarity with reported sequences of fungal species. The BLAST searches were run excluding uncultured/ environmental samples in the database. To corroborate the results, the BLAST search was repeated limiting the search to sequences only from type material. All sequences have been deposited in the GenBank database under the accession numbers that appear in Supplementary Table S1.
Morphological identification. Two species that did not have a close match to anything in Genbank, were examined in more detail to determine their morphological characteristics. Morphological analyses followed recommendations and techniques described by Ellis (1971) for hyphomycetous fungi and common methods in mycology 21,22 . Fungal isolates were cultured in CMD (BBL Corn Meal Dextrose agar, BD Company, France) and PDA (Difco Potato Dextrose agar, BD company, France) for 7 to 10 days near 25 °C. An optical microscope (Olympus BX-40, Japan) was used with an attached camera (18 megapixels, OMAX, Korea); software (ToupView, ToupTek Photonics, China) was used to measure structures.
Screening of cellulolytic activity. Cellulase-producing microorganisms were screened on agar plates enriched with only CMC as a source of carbon, with Gram's iodine as indicator (Prelab) [23][24][25][26] . This qualitative determination is based on the interaction of iodine with cellulose and its components in its degraded form, such that the integral biopolymer holds Gram's iodine dye; whereas areas with cellulose hydrolyzed by enzymes result in clear zones or the appearance of a pale halo 15,27 . The halo was measured for the subsequent calculation of the enzymatic index (EI), a semi-quantitative estimate of the enzyme activities, according to this formula 15 .

Results and Discussion
Isolation and identification of fungi isolated from drawings and lithographs. Through the screening of the lithographs, the total count of fungi isolated was 19, of which eight grew directly in water agar with CMC-km and eleven were first isolated from PDA and then recultivated in water agar with CMC as the sole source of carbon. The proliferation of fungi in the latter culture medium is in accordance with the environment in which they were isolated (limited sources of carbon, with cellulose as sole nutrient). Laminae #5 was the most contaminated, with ten isolations; followed by laminae #7 and #10, with 3 isolations each (see Supplementary Figure S1). The fungal isolates showed diverse forms, sizes, elevations, borders, surfaces, opacity, color and growth rates, as shown in Fig. 1.
BLAST searches in GenBank database resulted in the classification of nineteen isolates into fifteen species and nine genera (Table 1). These nineteen isolates had at least a 98% similarity with known species. The most prevalent genus was Cladosporium. Of the nine identified genera, Aspergillus, Chaetomium, Cladosporium, Penicillium, and Trichoderma are reported as common microbiota in ancient works of art 1,2,17,30,31 . The actin region was sequenced to confirm the results obtained with the ITS region, and to classify to species level some samples that could not be done with ITS. For all cases in which both ITS and actin sequences were obtained, the fungi were classified within the same species, except isolate #9 in which the actin region denied conclusive results obtained with ITS.
Two isolates were only identified to genus or class levels using both ITS and actin regions. Specifically, isolate #19 was classified within the genus Periconia, and isolate #21 was classified within the class Sordariomycetes, both in the phylum Ascomycota. Since these two isolates did not have a close match to any sequence in the Genbank, traditional morphological analyses and descriptions (e.g. microscopy and use of taxonomic literature) were done to elucidate the identity of these isolates.  Diagnosis: Nodulisporium-like conidiophore, with macro-and microconidia, hyaline, macroconidia 5-7-septate slightly curved, fusiform, microconidia cylindrical 1-2-septate. Colonies: At 25 °C after 3 weeks on CMD, reaching 20 mm diam., hyaline to white. On PDA attaining 25 mm diam., colony white, then turning purple (similar to OAC555), cracking and turning the media dull orange (lighter than OAC789). Conidiophores: Nodulisporium-like. . Notes: This species, because of the Nodulisporium-like conidiophore, is similar to Coniochaeta ershadii, especially in the size of the conidiogenous cells. The conidia produced by C. ershadii are prominently smaller 33 than those present in C. cipronana; the presence of macro-and microconidia is also a distinguishing character. The new fungal species described belong to Periconia Tode and Coniochaeta (Sacc.) Cooke genera (see Supplementary Figures S2 and S3). Periconia is a polyphyletic genus Pleosporales (Dothideomycetes, Ascomycota), with a complicated taxonomy and a poorly understood phylogeny 32 . This genus has been widely reported as a common endophyte from the roots of several plants, like a Periconia species isolated from Piper longum producing metabolites with a high pharmacological potential 34 and the melanized hyphae are believed to protect the fungi from environmental oxidation 35 . Some species have been reported as parasites in leaves of Xanthium strumarium and Ipomea muricara in India and others as decomposers in bamboo statches 36 . Coniochaeta (Coniochaetaceae, Coniochaetales, Sordariomycetes, Ascomycota) was introduced as a subgenus of Rosellinia De Not. for species with hairy perithecia but differing by the absence of amyloid asci in their sexual stages 33   Cellulase activity of the fungal isolates. Assay of the cellulase activity showed that 95% of the samples produce extracellular enzymes that break down cellulose into smaller oligosaccharides or monosaccharides, as evident from the clear zone observed after staining the plates with Gram's iodine (see Table 2 and Supplementary Figure S4). This fraction that includes the two new species (sample #19: Periconia epilithographicola and sample #21: Coniochaeta cipronana) also comprehends species of Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Penicillium, and Trichoderma, being the first four commonly reported with cellulolytic activity 31,[39][40][41][42][43][44][45] . These observations are congruent with the habitat of restricted carbon sources, in which sheets or laminae made of fibers of cellulose pulp were the support material for the growth of microorganisms.
Importantly, 32% of the total isolates had a significantly superior enzymatic index relative to a positive control (P. ostreatus), i.e., isolates #5 (Penicillium chrysogenum), #7 (Cladosporium tenuissimum), #11 (Cladosporium angustisporum) and #23 (Trichoderma cf. longibrachiatum). Other studies have characterized these species as effective cellulase producers 16,40 . Isolates #5 (Penicillium chrysogenum) and #23 (Trichoderma cf. longibrachiatum) had an outstanding performance relative to the positive control and the rest of the isolated fungi. Specifically, isolate #5 presented an enzymatic index for cellulose activity almost twice of that of the positive control. Isolates of these species not only have presented important cellulase activity but also have been the object of study for their capacity to produce xylanases 46 , or tanases 47 .
The case of isolate # 23 (Trichoderma cf. longibrachiatum) was even more striking. For this fungus, EI is reported for 24 h (see Table 2) because after 7 days (the period in which the other isolates were measured) the microorganism had covered the entire Petri plate, evidence of an accelerated growth and a large capacity to use the CMC as the sole source of carbon. The result (1.39 ± 0.03) was slightly smaller than the positive control (1.8 ± 0.1, measured after seven days). However, as previously mentioned, isolate # 23 was measured at 24 h. This result implies a large rate of enzymatic (cellulase) production from fungus #23 in a medium rich in cellulose, relative to the rest of the fungi studied, which is important for the development of biotechnological applications and industry. Many studies have featured this species as a fungus with great cellulase activity [48][49][50][51] . Many commercial cellulases can be purchased in purified form after production with this species (e.g. C9748 Sigma-Aldrich or E-CELTR from Megazyme). Investigations with isolation # 23 will continue to evaluate its potential to degrade lignocellulosic residues from agricultural activity in Costa Rica (e.g., wastes from pineapple production).
In summary, in isolating, identifying and characterizing the cellulolytic activity of the fungi responsible for the biodegradation of a nineteenth-century collection, several species of fungi were found to have the ability to produce cellulases. In addition, two new species of fungi were identified and named Periconia epilithographicola sp. nov. and Coniochaeta cipronana sp. nov., which also have cellulolytic activity. A knowledge of the microorganisms that colonized the Bernard Romain Julien collection belonging to Universidad de Costa Rica will allow the development of strategies directed to the conservation of these ancient lithographs. This work also contributes to the knowledge of new species with cellulolytic activity, which is a topic of perennial interest for biotechnology because of the important role of fungal cellulolytic enzymes in commercial food processing, performing the hydrolysis of cellulose during drying of beans, in the textile industry and laundry detergents, in the conversion of biomass into industrially important solvents or fuels, and their potential application for the bioremediation of wastes.