Fungi with history: Unveiling the mycobiota of historic documents of Costa Rica 1 2 Efraín Escudero-Leyva1,2, Sofía Vieto1, Roberto Avendaño1, Diego Rojas-Gätjens1, Paola 3 Agüero3, Carlos Pacheco4, Mavis L. Montero3,5, Priscila Chaverri2,6* & Max Chavarría1,2,3* 4 5 1Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), CeNAT-CONARE, 1174-1200 6 San José, Costa Rica. 2Centro de Investigaciones en Productos Naturales (CIPRONA), 7 Universidad de Costa Rica, 11501-2060 San José, Costa Rica. 3Escuela de Química, Universidad 8 de Costa Rica, 11501-2060 San José, Costa Rica. 4Archivo Nacional de Costa Rica, San José, 9 Costa Rica. 5Centro de Investigación en Ciencia e Ingeniería de Materiales (CICIMA), 10 Universidad de Costa Rica, 2060 San Pedro, San José, Costa Rica. 6Escuela de Biología, 11 Universidad de Costa Rica, 11501-2060 San José, Costa Rica. 12 13 14 *Corresponding authors: 15 Priscila Chaverri (priscila.chaverriechandi@ucr.ac.cr) 16 Escuela de Biología and Centro de Investigaciones en Productos Naturales, Universidad de Costa 17 Rica, 11501-2060 San José, Costa Rica. 18 Max Chavarría (max.chavarria@ucr.ac.cr) 19 Escuela de Química and Centro de Investigaciones en Productos Naturales, Universidad de Costa 20 Rica, 11501-2060 San José, Costa Rica. 21 22 23 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Abstract 24 25 Through nondestructive techniques, we studied the physicochemical characteristics and mycobiota 26 of five key historic documents from Costa Rica, including the Independence Act of Costa Rica 27 from 1821. We determined that for documents dated between 1500 and 1900 (i.e., the Cloudy Days 28 Act, the Independence Act, and two documents from the Guatemalan Series from 1539 and 1549), 29 the paper composition was cotton, whereas the 1991 replicate of the Political Constitution from 30 1949 was made of wood cellulose with an increased lignin content. We also determined that the 31 ink employed in 1821 documents is ferrogallic, i.e., formed by iron sulfate salts in combination 32 with gallic and tannic acids. In total, 22 fungal isolates were obtained: 15 from the wood-cellulose-33 based Political Constitution and seven from the other three cotton-based documents. These results 34 suggest that cotton-based paper is the most resistant to microbial colonization. Molecular 35 identifications using three DNA markers (i.e., ITS nrDNA, beta-tubulin, and translation elongation 36 factor 1-alpha) classified the isolates in eight orders and ten genera. The most frequent genera were 37 Cladosporium, Penicillium, and Purpureocillium. Of the isolates, 95% presented cellulolytic 38 activity correlated to their ability to cause deterioration of the paper. This work increases the 39 knowledge of the fungal diversity that inhabits historic documents and its relationship with paper 40 composition and provides valuable information to develop strategies to conserve and restore these 41 invaluable documents. 42 43 Keywords 44 Biodeterioration; historic documents of Costa Rica, Independence Act of Costa Rica, 45 Cladosporium, Penicillium, Purpureocillium, cellulolytic activity. 46 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Introduction 47 48 Because biodeterioration can lead to the damage of historic documents, artwork, 49 monuments, or buildings, its study is fundamental for the conservation of cultural heritage 50 (Sterflinger and Pinar 2012; Palla and Barresi 2017; Vieto et al. 2021; Ranalli and Zanardini 2021; 51 Ranalli et al. 2005). The prevention of biodeterioration and development of adequate conservation 52 and restoration strategies cannot be an unscripted process; it is necessary to undertake diagnoses 53 of these valuable pieces of our history and art, which include chemical characterization and the 54 study of microbial diversity together with the physiological characteristics of biodeteriogens (Palla 55 and Barresi 2017; Negi and Sarethy 2019). 56 Valuable cultural and historic objects, such as relevant paintings, ancient sculptures, and 57 historic documents, can be seen as substrates on which microorganisms can thrive and cause 58 damage. Specifically, paper-based documents contain biodegradable organic constituents that 59 fungi can use as a substrate (Jia et al. 2020; Pyzik et al. 2021). The term “paper” is a general 60 concept that encompasses all thinly laminated material that is produced with vegetable fiber pulp 61 or other materials ground and mixed with water, dried, and hardened. Historically, vegetable fibers 62 have been extracted from natural sources, such as straw, silk, hemp, flax, cotton, and the bark of 63 different trees, among others. The content of cellulose and other components of paper can vary 64 depending on its origin, generating papers that are more or less resistant to biodegradation as a 65 consequence. For example, it is well known that cellulose fibers have high purity in cotton and 66 linen papers, which results in papers with greater durability and resistance to biodeterioration 67 (Negulescu et al. 1998; Daria et al. 2020). 68 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Damage produced by fungi that is normally present on paper —including staining, 69 material weakening, and partial or complete destruction of documents— can occur in the long term 70 (Sequeira et al. 2019). Besides the alterations caused to the documents, the health of curators and 71 people involved in archives or museums can also be threatened if the spore production is elevated 72 or mycotoxins are produced (Sterflinger and Pinzari 2012). The study of fungi responsible for the 73 biodegradation of paper began in 1818 with the pioneering work of Christian Gottfried Ehrenberg 74 (Sterflinger and Pinzari 2012). To date, diverse fungi have been identified in old paperwork, such 75 as Aspergillus, Chaetomium, Cladosporium, Penicillium, and Trichoderma; occasionally, new 76 species can even be found (Coronado-Ruiz et al. 2018; Sequeira et al. 2019). Mesquita et al. (2009) 77 isolated, identified, and characterized the microbiota from historic documents dated between 78 1860–1939 in the archive of the University of Coimbra and found fourteen fungal genera, of which 79 Aspergillus, Cladosporium, and Penicillium were the most common. Our research group recently 80 isolated nineteen fungi from a nineteenth-century French collection of drawings and lithographs 81 in the custody of Universidad de Costa Rica (Coronado-Ruíz et al. 2018). The fungi were 82 molecularly identified as Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, 83 Penicillium, and Trichoderma; a great majority of them showed cellulolytic activity. Many fungal 84 species found in historic paper-based documents contain enzymatic activity related to 85 biodeterioration, which allows fungi to use these surfaces as a source of carbon (Coronado-Ruíz 86 et al. 2018; Pinheiro et al. 2019; Vieto et al. 2022). The enzymatic machinery to take advantage 87 of paper as a source of carbon has been reported in fungi isolated from historic documents and 88 includes the presence of exoenzymes with cellulase activity (Puškárová et al. 2019; El Begardi et 89 al. 2014), lignocellulolytic (Mazzoli et al. 2018) glucanase, and laccase (Sterflinger and Pinzari 90 2012). 91 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ The National Archive of Costa Rica (NACR) —called Archivos Nacionales (National 92 Archives) before 1948— is where the most treasured documents in Costa Rica are preserved; these 93 include the Cloudy Days Act (Acta de los Nublados, September 28, 1821), in which authorities of 94 the Municipality of León, in the Captaincy General of Guatemala, expressed their position on 95 Central American Independence; the Political Constitution with all historic changes, including the 96 abolition of the Costa Rican army (Jaén García 2019; Chacón León 2021); and perhaps the most 97 important historic document in the country: the Costa Rican Independence Act (Acta de 98 Independencia, October 29, 1821). These invaluable documents are in addition to more than 99 20,000 linear meters of other paper-based documents that contain the history of Costa Rica and 100 that are in the custody of NACR. Due to the tropical peculiarities of the country —such as high 101 humidity, heat, long rainy seasons, and sometimes inadequate storage conditions— documents and 102 artworks are constantly threatened and come under continuous biodeterioration, making the 103 conservation of the country’s cultural heritage a challenge (Silva, 2011). 104 The aim of this work was to characterize the paper composition and evaluate the presence 105 and cellulolytic activity of culturable fungi in historic documents from the National Archive of 106 Costa Rica, including invaluable documents such as the Independence Act of Costa Rica. This 107 information enables restorers to establish guidelines for the preservation and restoration of paper-108 based historic documents. 109 110 Materials and Methods 111 112 Sampling of documents 113 114 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Permits to sample the historic documents were obtained from the Institutional Commission 115 of Biodiversity of the University of Costa Rica (resolution Nº 186) and authorities of the NACR. 116 Historic documents stored at NACR were sampled between March and September 2019. The 117 documents were: (i) Political Constitution redacted in 1949 (1991 replicate), (ii) Cloudy Days Act 118 from 1821 (Acta de los Nublados), (iii) Independence Act of Costa Rica from 1821, and (iv) two 119 documents from the Guatemalan Series from 1539 and 1549 (Fig. 1). For fungal isolation, careful 120 rubbing with sterile cotton swabs over the surface of the documents was performed, especially 121 seeking signs of biodeterioration such as dark spots. The swabs were then saved inside Falcon 122 tubes for transport to the laboratory. 123 124 Material characterization by attenuated-total-reflection Fourier-transform infrared spectra (ATR-125 FTIR). 126 127 ATR-FTIR was used to determine functional groups and distinguish cellulosic materials. 128 These ATR-FTIR spectra were recorded using a portable spectrophotometer (Bruker Alpha II, 129 Canada) with platinum ATR mode and monolithic diamond crystal. The spectral resolution was 4 130 cm-1, in wavenumber range 400–4,000 cm-1 with 99 scans. To carry out the identification, the 131 “Database of ATR-FT-IR spectra of various materials” (Vahur et al. 2016) was used. 132 133 Material characterization by X-ray fluorescence (XRF). 134 135 X-ray fluorescence (XRF) was used to determine the elemental composition of the 136 material, especially the presence of metallic ions, such as iron and calcium, among others. This 137 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ technique is especially important in the characterization of inks. The XRF spectra were recorded 138 with a portable XRF spectrophotometer (Elio, XGLab; Bruker, Italy) measured at the Ka line of 139 manganese (resolution 140 eV), a SDD detector (active area 25 mm2, fluorescence angle 63°, 140 incident angle 90°) and a distance of 14 mm from the detector to the sample. The electric current 141 was adjusted to 80 µA, with a voltage of 50 kV and a measuring duration of 300 s. Software XRS-142 FP2 (CrossRoads Scientific, USA) was used for data analysis, maintaining a noise signal of 0.5. 143 144 Fungal cultivation strategy 145 146 Samples were processed in the laboratory 2-4 hours after sampling. Cotton swabs were 147 immersed into sterile phosphate-buffered saline solution (PBS; 400 µL, 1X; Thermo Fisher 148 Scientific, USA) and homogenized using a vortex (40 s). Each sample (100 µL) was then cultured 149 in plates of potato dextrose agar (1% Difco PDA; BD company, France) and carboxymethyl 150 cellulose (1% CMC; Sigma Aldrich, USA, with 0.8 % agar; BD company, France) with kanamycin 151 (50 µg/mL; Sigma-Aldrich, USA) and incubated (25 °C) until growth was observed. Colonies 152 exhibiting varied morphologies were purified and transferred onto PDA plates; photographs were 153 taken after incubation (15 days, 30 °C). 154 155 Molecular identification of the isolated fungi 156 157 Genomic DNA was extracted from the isolated fungi using the method described by Lodhi 158 et al. (1994) with modifications. First, two agar disks (diameter 8 mm) from each fungus were 159 added to a centrifuge tube (2 mL) and were ground with sterile micro-pestles. Extraction buffer 160 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ (750 µL, sodium EDTA 20 mM, tris-HCl 100 mM, NaCl 1.4 M, CTAB 2 % [w/v], PVP 2% [w/v] 161 and ß-mercaptoethanol 0.2%) was added; the tubes were vortexed and incubated (20 min, 65 °C). 162 For DNA separation, trichloromethane-octanol (750 µL, 24:1) was added to the mixture and 163 centrifuged (25 °C, 14,000 rpm). DNA from the top aqueous phase (600 µL) was precipitated on 164 an addition of 2-propanol (600 µL; Sigma-Aldrich, USA). Then, ethanol (70 %, 500 µL; Sigma-165 Aldrich, USA) was used to wash the precipitated DNA. Finally, DNA was resuspended in Tris-166 EDTA buffer (50 µL) with RNAse (1 µL, 10 mg/mL; Thermo Fisher Scientific, USA). 167 To obtain a preliminary identification of the isolates, the nrDNA internal transcribed 168 spacers (ITS) were amplified with primers ITS4 (TCCTCCGCTTATTGATATGC) and ITS5 169 (GGAAGTAAAAGTCGTAACAAGG) (White et al.1990). Depending on the results from ITS, 170 secondary markers were used to refine the identifications for some isolates, i.e., portions of the 171 translation elongation factor 1-alpha (TEF1; primers CATCGAGAAGTTCGAGAAGG and 172 TACTTGAAGGAACCCTTACC) (Carbone and Kohn 1999) and beta-tubulin (TUB2; primers 173 AACATGCGTGAGATTGTAAGT and TAGTGACCCTTGGCCCAGTTG) (O'Donnell and 174 Cigelnik 1997) genes. Each reaction (total volume 20 µL) consisted of Master Mix (10 µL, 2X; 175 Thermo Fisher Scientific, USA), bovine serum albumin (BSA, 0.5 µL, 20 mg/mL; Sigma Aldrich, 176 USA), dimethyl sulfoxide (DMSO, 1.5 µL; Sigma Aldrich, USA), and primers (0.5 µL each, 10 177 µM) and DNA (2 µL, 50 ng/µL). PCR reactions were implemented in a thermal cycler (9902 178 Veriti, Applied Biosystem, Norwalk, USA), according to conditions described by Schoch et al. 179 (2012) for ITS, Carbone and Kohn (1999) for TEF1 and O'Donnell and Cigelnik (1997) for TUB2. 180 Sanger sequencing of PCR products was performed with Psomagen (USA); the raw sequences 181 were edited and assembled in Bioedit v.7.2. Isolate identification was performed by comparing the 182 consensus sequences against the GenBank database using the BLAST search tool. Then, a 183 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ cladogram was constructed using the ITS sequences. For this, the two closest matches, with type 184 material prioritized, were retrieved/downloaded from the BLAST analysis and aligned using 185 MUSCLE (Edgar 2004). The resulting alignment in Phylip format was submitted to Bayesian 186 Inference analysis with Exabayes (Aberer et al. 2014). MCMC was run in parallel and 15 million 187 generations were done with 25% burn-in. All analyses were run in the Kabré supercomputer 188 (CNCA-CONARE, Costa Rica). A consensus tree was visualized and edited with FigTree v.1.4.3 189 (Rambaut 2010). Newly generated sequences were deposited in GenBank under accession 190 numbers ON479855-ON479876 (ITS), ON720280-ON720285 (TEF1), and ON734081– 191 ON734096 (TUB2). 192 193 Screening of cellulolytic activity 194 195 The screening of cellulase-producing fungi was undertaken on carboxymethyl 196 cellulose plates (CMC, 1 %; Sigma Aldrich, USA) as the sole carbon source, supplemented with 197 agar (0.8 %, Sigma Aldrich, USA; Johnsen and Krause 2014; Gohel et al. 2014). For this purpose, 198 agar disks (diameter ~0.8 mm) of each fungus were placed in the center of CMC plates and 199 incubated (7 days, 30 °C). After incubation, each plate was flooded with Gram’s iodine stain (10 200 mL; Sigma-Aldrich, USA; Kasana et al. 2008; Gohel et al. 2014) and washed with water for 10 201 min. Because Gram’s iodine dye is held only by integral cellulose polymers, cellulase activity is 202 revealed by the clear zones appearing as pale halos (Florencio et al. 2012). Photographs were taken 203 before and after staining the plates; software ImageJ v.1.52k (Bourne 2010) was used to measure 204 fungal growth (as the diameter of the colony) and the halo diameter for a subsequent calculation 205 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ of the enzymatic index (EI), a semiquantitative estimate of enzyme activity according to the 206 following formula (Florencio et al. 2012): 207 208 EI = Diameter of hydrolysis zone Diameter of colony 209 210 The experiments were performed in triplicate; Pleurotus ostreatus served as a positive control 211 (Garzlllo et al. 1994; Valášková and Baldrian 2006). 212 213 Results 214 215 Material characterization by infrared spectra (ATR-FTIR) and X-ray fluorescence (XRF) 216 217 Macroscopically all documents analyzed showed detailed damage as observed in Fig. 1, in 218 which signs of humidity and possible leakage are visible in several areas. Particularly, in the 219 Independence Act surface, orangish spots were present. When observed under UV light, those 220 areas are fluorescent and appear as dark spots in UV reflectance photographs (Supplementary Fig. 221 S1). 222 The chemical composition of historic documents (both inks and paper) was studied through 223 nondestructive and portable techniques (See Materials and Methods). The composition of each 224 document is shown in Table 1. The composition of the organic substrate was determined by 225 comparing the IR spectra with databases; the elements present in the ink and additives were 226 resolved with X-ray fluorescence. The results showed that the paper in the documents from 1500–227 1900 was handmade mainly from cotton with watermark presence (excepting the second page from 228 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ the Cloudy Days Act, which was cellulose-based). In the Political Constitution (1991 replicate), 229 modern paper was used, characterized by shorter fibers and greater lignin content; this indicates 230 that this paper was made from wood cellulose. The results showed that the ink employed in the 231 documents from 1821 was ferrogallic, formed by iron sulfate salts in combination with gallic and 232 tannic acids (Table 1 and Supplementary Fig. S1). 233 234 Isolation and identification of fungi 235 236 In total, 22 fungal isolates (Supplementary Fig. S2) were recovered from the Costa Rican 237 historic documents, being the Political Constitution (1991 replicate) that with the most isolates (15 238 in total). The taxonomic identification provided by BLAST and ITS phylogenetic analyses are 239 shown in Table 2 and Fig. 2, respectively. The fungi recovered belong to 14 genera and eight 240 orders. Two taxa were obtained from the Independence Act, two from the Cloudy Days Act, two 241 from the document from 1549, and only one fungal isolate from the oldest document (Guatemalan 242 Series 1539). The phylogenetic placement of the isolates corresponded to eight orders, as shown 243 in the ITS cladogram (Fig. 2). The phylogenetic analysis supports the identifications performed 244 with the BLAST tool, at least at the genus level. TEF1 and TUB2 sequences refined the 245 identification for some of the isolates (Table 2). 246 Most of the fungi found belong in the Ascomycota (86%), followed by Basidiomycota 247 (14%). Among the resulting orders, the majority belong in Hypocreales (23%; Acremonium, 248 Beauveria, and Purpureocillium), Eurotiales (18%; Aspergillus and Penicillium), and Capnodiales 249 (18%; Cladosporium). The Basidiomycota was represented by Coprinellus, Trametes and an 250 unidentified species of Psathyrellaceae. 251 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ 252 Cellulolytic activity 253 254 The results from cellulolytic activity are shown in Table 3. For the 22 isolates tested, the 255 Enzymatic Index (EI) average was 2.45, with Cyphellophora aff. pluriseptata CP2-A4P isolated 256 from the Political Constitution being the fungus with the greatest cellulolytic activity (4.0 ± 0.3), 257 followed by Penicillium steckii ND1-A1P (3.3 ± 0.3), and Cladosporium sp. CP1-A2P (3.3 ± 0.1). 258 In contrast, Purpureocillium lilacinum 1539-A1P —from the 1539 Guatemalan Series— was the 259 only isolate without cellulolytic activity. The other fungal isolates showed cellulolytic activity 260 above the levels of the control (Pleurotus ostreatus), except for Trametes CP1-A3C. 261 262 Discussion 263 264 In this work we determined the chemical and microbiological composition of five 265 important historic documents of Costa Rica, including the Independence Act of 1821. Through 266 spectral techniques, we determined that for the documents dated between 1500 and 1900 (i.e., the 267 Cloudy Days Act, the Independence Act of Costa Rica of 1821, and two documents from the 268 Guatemalan Series of 1539 and 1549), the composition of the paper was cotton (i.e., approximately 269 90% cellulose (Felgueiras et al. 2021), except the second page of the Cloudy Days Act, which is 270 composed of cellulose acetate. The 1991 replicate of the Political Constitution of 1949 was made 271 of cellulose and lignin; this paper presented the greatest amount of lignin, which indicates a modern 272 paper made from wood (hereafter referred to as wood cellulose-based paper). 273 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Despite that modern paper is relatively stable, deterioration is common with increased 274 levels of humidity and acidification produced by oxidation and the presence of microorganisms 275 (Proniewicz et al. 2001). Although the 1991 replicate of the Political Constitution from 1949 is 276 the most recent, 15 fungal isolates were obtained, whereas from all the oldest documents (made 277 mainly of cotton), seven in total were obtained. These data suggest that wood-cellulose-based 278 paper possesses characteristics that are more suitable for fungal colonization than cotton-based 279 documents. These observations make sense if we consider the differences between the chemical 280 composition of cotton and paper obtained from other plant fibers such as wood. Cotton contains 281 approximately 90% cellulose (hence it is sometimes referred to as highly pure cellulose), whereas 282 other natural fibers, such as wood, contain 40–55% cellulose combined with other constituents 283 such as lignin and hemicelluloses (Felgueiras et al. 2021). Cellulose is considered a two-phase 284 material, having both crystalline and amorphous phases (Ling et al. 2019). Cotton cellulose fibers 285 are reported to have a greater degree of polymerization and crystallinity, which generates stronger 286 fibers and greater resistance to hydrolysis and biodegradation. (Itävaara et al. 1999). Therefore, 287 these two characteristics (higher cellulose content and higher crystallinity) make cotton a substrate 288 that is less prone to microbial colonization than a material such as wood-based paper, which is the 289 case of the paper of the 1991 replicate of the Political Constitution of 1949. Enzymatically, the 290 reduced ability for microbial colonization of cotton-based papers is related to the more limited 291 access of the cellulase enzyme complex to the substrate due to the orderly and compact architecture 292 of the crystalline cellulose present in cotton (Arantes and Saddler 2010). 293 In Fig. 1C, the orangish spots over the Independence Act surface indicate oxidation from 294 cellulose and iron, probably caused by both abiotic and biotic factors (Choi 2007). Those areas are 295 fluorescent under UV light and appear as dark spots in UV reflectance photographs 296 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ (Supplementary Fig. S1). Although excessive humidity can itself trigger oxidation, contributing to 297 the damage of important documents, the inks used can also be affected by this abiotic factor, 298 producing the migration of metal ions of common ink components such as iron and copper, 299 compromising the preservation of historic and cultural heritage due to weakened paper (Henniges 300 et al. 2006). Our work determined that the ink employed in the documents from 1821 was 301 ferrogallic, formed by iron sulfate salts in combination with gallic and tannic acids. The last was 302 confirmed from the XRF and multispectral photography (see Supplementary Fig. S1). Ink of this 303 kind is visible under infrared and, in darkness, under UV light (Havermans et al. 2003). The 304 implementation of optical spectroscopy techniques, such as those used in this work, have been 305 shown to help identify the early stages of document damage by microorganisms such as fungi, 306 relating changes in the spectral composition to the active presence of fungi (Povolotckaia et al. 307 2019). 308 In the historic documents, we obtained a total of 22 fungi belonging to 14 genera, of which 309 five (35%) were previously identified in paper-based historic documents (Bensch et al. 2018; 310 Pinheiro et al. 2019; Romero et al. 2021; Trovão and Portugal 2021). The presence of two 311 Chaetothyriales members (isolates 1549-4A1C and CP2-A4P) is interesting because they are 312 inhabitants of environments with limited resources, such as rocks, insects, and ant nests; some 313 species in the order can even become pathogenic for humans, especially in tropical regions ( Attili-314 Angelis et al. 2014; Ahmed et al. 2021). Cyphellophora, also Chaetothyriales, is closely related to 315 Phialophora (Feng et al. 2014), which includes species that grow in extremely acidic conditions 316 and have been reported to produce ß-mannanase and ß -glucanase enzymes (Zhao et al. 2010; Zhao 317 et al. 2012). Our isolate CP2-A4P had the greatest enzymatic index value (4.0 ± 0.3), for which 318 further analysis involving enzyme identification could yield intriguing results. Isolate Penicillium 319 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ steckii ND1-A1P originated from a cotton-based substrate and presented an enzymatic index of 320 3.3 ± 0.3. This corresponds to what is commonly observed because Aspergillus and Penicillium 321 species are registered continuously in paper biodeterioration and are known to break the hydrogen 322 bonds, which translates into a weakening of the documents, regardless of their composition 323 (Povolotckaia et al. 2019). In total, three isolates corresponding to Penicillium were obtained from 324 the Cloudy Days Act, the Political Constitution, and the 1549 Guatemalan Series. We registered 325 only a single isolate corresponding to Aspergillus hiratsukae (AI3-A1P), which was recovered 326 from the Independence Act of 1821. This was unexpected because Aspergillus spp. are frequent 327 biodeteriogens in cultural heritage objects and spaces in which historic documents are preserved, 328 such as the National Archive of Cuba, in which Aspergillus, Cladosporium, and Penicillium were 329 the most frequent airborne genera reported (Borrego and Perdomo 2016). In this work, 330 Cladosporium isolates were present in the Cloudy Days Act (isolate ND2-A1P), the Independence 331 Act (isolate AI1-A1C), and the Political Constitution (isolates CP1-A1P and CP1-A2P), 332 corresponding to cellulose acetate, cotton, and wood cellulose substrates. Cladosporium spp. are 333 reported as colonizers in agricultural waste (Herculano et al. 2011), artworks (Coronado-Ruiz et 334 al. 2018), repositories of historic documents (Borrego and Perdomo 2016), and as extremophiles 335 in high-altitude tropical glaciers (Calvillo-Medina et al. 2020). 336 Neopestalotiopsis clavispora (CP1-A2C) and Pestalotiopsis kenyana (CP2-A2C) 337 (Xylariales), both obtained from the Political Constitution, belong to a genus known as a common 338 endophyte, saprotroph, and phytopathogen in diverse plants and climates (Reddy et al. 2016). 339 Because of the various ecological relationships this genus has with plants, it has attracted attention 340 for its cellulolytic abilities, with over 400 possible enzymes found through the study of a 341 Pestalotiopsis isolated from a mangrove (Arfi et al. 2013). So far, there are some cellulolytic 342 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ enzymes known from this genus that have been studied, such as the xylanase (Koh et al. 2021) or 343 the cellulases (Goukanapalle et al. 2020). Another single isolate obtained from the Political 344 Constitution was Periconia CP1-A1C, which belongs to the dark septate fungi Pleosporales, with 345 some isolates reported with thermostable b-Glucosidases suitable for biotechnological processes 346 (Harnpicharnchai et al. 2009). Periconia species have been encountered in extreme environments, 347 such as deserts, seas, tropical glaciers (Calvillo-Medina et al. 2020), and even a new species 348 growing over lithographs from the 19th century (Coronado-Ruiz et al. 2018), among others. 349 Hypocreales isolates were only found in the Political Constitution. Intriguing were two 350 isolates known as insect pathogens, i.e., Beauveria (CP2-A3P) and Purpureocillium (1539-A1P 351 and CP2-A1P; Shrestha et al. 2019). The cellulolytic activity recorded for our Beauveria isolate 352 was 3.1 ± 0.3, which is consistent with reports of cellulolytic activity driven by a thermally stable 353 b-Glucosidase in Beauveria bassiana (Borgi and Gargouri 2016). Screening for cellulolytic 354 activity in this entomopathogenic species is not commonly done because it is mainly studied for 355 its biological control abilities in multiple agricultural crops (Posada and Vega 2006; Sanjuan et al. 356 2014; Mwamburi 2021). Beauveria has also been found as an endophyte, along with 357 Purpureocillium (Kepler et al. 2013). An isolate of Purpureocillium lilacinum has been reported 358 as a biodeteriogen of indoor materials able to grow in alkaline materials, producing damage in 359 limestones and plasters of cultural heritage in Russia (Ponizovskaya et al. 2019). The tolerance to 360 extreme conditions by some Purpureocillium spp. results in the ability to become pathogenic to 361 humans and resistant to fungicides (Calvillo-Medina et al. 2020). Although one isolate recovered 362 from the 1539 Guatemalan Series (cotton-based) was unable to grow in the carboxymethyl 363 cellulose media, isolate CP2-A1P from the Political Constitution (wood cellulose-based) attained 364 EI of 3.2. Acremonium isolates CP2-A3C and ND2-A1P registered enzymatic indexes < 3. 365 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Previous researchers reported a xylanase produced by Acremonium cellulolyticus (Watanabe et al. 366 2014); another group subsequently revised the taxonomy and concluded a misidentification of an 367 Acremonium species, reidentifying isolate Y-94 from Japan as Talaromyces (Eurotiales) (Fujii et 368 al. 2014). Apart from the latter, only a xylanase has been reported for A. alcalophilum (Šuchová 369 et al. 2020). 370 Small enzymatic indexes also occurred for three basidiomycetes recovered from the 371 Political Constitution (CP1-A3C, CP1-A3P, and CP2-A2P). These were identified as belonging to 372 Psathyrellaceae, including Coprinellus (CP2-A2P). From these, only one report exists of a 373 xylanase produced by Coprinellus disseminatus, which was tolerant to varied pH and temperatures 374 (Agnihotri et al. 2010). Isolate Trametes CP1-A3C registered an EI less than the control (Pleurotus 375 ostreatus). Species with tough fruiting bodies, such as Trametes maxima, are reported to possess 376 laccase activity, even when exposed to herbicides (Cupul et al. 2014); an isolate of T. versicolor 377 is reported to cause the effective degradation of fungicides (Rodríguez-Rodríguez et al. 2012). 378 379 Conclusions 380 381 Regardless of the material of a document of origin —cotton or wood cellulose— most 382 recovered fungal isolates presented cellulolytic activity. From the historic documents sampled, the 383 Political Constitution had the greatest number of isolates, which suggests that wood cellulose-384 based paper possesses characteristics more suitable for fungal colonization than the oldest cotton-385 based documents (i.e., documents from 1500–1900). Cotton contains 90% cellulose and has great 386 crystallinity, which makes it more difficult for the enzymatic machinery of microorganisms to 387 degrade these polymers and use them for their nutritional requirements. Even though the oldest 388 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ documents (e.g., Independence Act and the Cloudy Days Act) yielded few isolates, restoration and 389 improvement of the conditions they are stored in should be implemented to avoid oxidation and 390 weakening of their fibers which could then increase microbiological contamination. The results of 391 our work provide valuable information for establishing the appropriate protocols to undertake that 392 restoration and conservation. Determining the chemical composition of the paper and the 393 composition of the inks, as well as the microbiological load, allows for identification of the most 394 appropriate strategies and treatments to restore documents as important to Costa Rica as the Act 395 of Independence itself. Because historic documents can be considered microhabitats with limited 396 resources, the screening of species with novel biotechnological applications in such environments 397 is a promising and fascinating field. The study of how best to conserve historic documents is vital 398 to preserve, in a satisfactory condition, important sources and records of human history. 399 Multidisciplinary approaches such as the present work can help curators make the best choice of 400 restoration techniques and eventually fulfill the Korean phrase: “Silk can stand five hundred years, 401 but paper can stand one thousand” (Jeong et al., 2014). 402 403 References 404 Aberer, A.J., Kobert, K. and Stamatakis, A. (2014) ExaBayes: massively parallel Bayesian tree 405 inference for the whole-genome era. Mol Biol Evol 31, 2553–2556. 406 407 Agnihotri, S., Dutt, D., Tyagi, C.H., Kumar, A. and Upadhyaya, J.S. (2010) Production and 408 biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from 409 Coprinellus disseminatus SW-1 NTCC 1165. World J Microbiol Biotechnol 26, 1349–1359. 410 411 Ahmed, S.A., Bonifaz, A., González, G.M., Moreno, L.F., Menezes da Silva, N., Vicente, V.A., 412 Li, R. and de Hoog, S. (2021) Chromoblastomycosis caused by Phialophora-proven cases from 413 Mexico. J Fungi 7, e95. 414 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ 415 Arantes, V. and Saddler, J.N. (2010) Access to cellulose limits the efficiency of enzymatic 416 hydrolysis: the role of amorphogenesis. Biotechnol Biofuels 3, e4. 417 418 Arfi, Y., Chevret, D., Henrissat, B., Berrin, J.G., Levasseur, A. and Record, E. (2013) 419 Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus 420 Pestalotiopsis sp. Nat Commun 4, 1810–1819. 421 422 Attili-Angelis, D., Duarte, A.P.M., Pagnocca, F.C., Nagamoto, N.S., De Vries, M., Stielow, J.B. 423 and De Hoog, G.S. (2014) Novel Phialophora species from leaf-cutting ants (tribe Attini). Fungal 424 Divers 65, 65–75. 425 426 Bensch, K., Groenewald, J. Z., Meijer, M., Dijksterhuis, J., Jurjević, Ž., Andersen, B., Houbraken, 427 J., Crous, P.W. and Samson, R.A. (2018) Cladosporium species in indoor environments. Stud 428 Mycol 89, 177–301. 429 430 Borgi, I. and Gargouri, A. (2016) A novel high molecular weight thermo-acidoactive β-glucosidase 431 from Beauveria bassiana. Appl Biochem Microbiol 52, 602–607. 432 433 Borrego, S. and Perdomo, I. (2016) Airborne microorganisms cultivable on naturally ventilated 434 document repositories of the National Archive of Cuba. Environ Sci Pollut Res, 23, 3747–3757. 435 436 Bourne, R. (2010). ImageJ. In: Fundamentals of digital imaging in medicine. Springer, London. 437 438 Calvillo-Medina, R.P., Gunde-Cimerman, N., Escudero-Leyva, E., Barba-Escoto, L., Fernández-439 Tellez, E.I., Medina-Tellez, A.A., Bautista-de Lucio, V., Ramos-López, M.Á., and Campos-440 Guillén, J. (2020) Richness and metallo-tolerance of cultivable fungi recovered from three high 441 altitude glaciers from Citlaltépetl and Iztaccíhuatl volcanoes (Mexico). Extremophiles 24, 625–442 636. 443 444 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Carbone, I. and Kohn, L.M. (1999) A method for designing primer sets for speciation studies in 445 filamentous ascomycetes. Mycologia 91, 553-556. 446 447 Chacón León, L.A. (2021) El bicentenario de la independencia de Costa Rica. Revista del Archivo 448 Nacional de Costa Rica 85, 1–13. 449 450 Choi, S. (2007) Foxing on paper: a literature review. J Am Inst Conserv 46, 137-152. 451 452 Coronado-Ruiz, C., Avendaño, R., Escudero-Leyva, E., Conejo-Barboza, G., Chaverri, P. and 453 Chavarría, M. (2018) Two new cellulolytic fungal species isolated from a 19th-century art 454 collection. Sci Rep 10, e7492. 455 456 Cupul, W.C., Abarca, G.H., Vázquez, R.R., Salmones, D., Hernández, R.G. and Gutiérrez, E.A. 457 (2014) Response of ligninolytic macrofungi to the herbicide atrazine: dose-response bioassays. 458 Revista Argentina de Microbiologia 46, 348–357. 459 460 Daria, M., Krzysztof, L. and Jakub, M. (2020) Characteristics of biodegradable textiles used in 461 environmental engineering: A comprehensive review. J Clean Prod 268, e122129. 462 463 Edgar, R.C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space 464 complexity. BMC Bioinform 5, 1-19. 465 466 El Bergadi, F., Laachari, F., Elabed, S., Mohammed, I.H. and Ibnsouda, S.K. (2014) Cellulolytic 467 potential and filter paper activity of fungi isolated from ancients manuscripts from the Medina of 468 Fez. Ann Microbiol 64, 815–822. 469 470 Felgueiras, C., Azoia, N.G., Gonçalves, C., Gama, M. and Dourado, F. (2021) Trends on the 471 cellulose-based textiles: raw materials and technologies. Front Bioeng Biotechnol 9, 608826. 472 473 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Feng, P., Lu, Q., Najafzadeh, M.J., van den Ende, A.H.G.G., Sun, J., Li, R., Xi, L., Vicente, V.A., 474 Lai, W., Lu, C. and de Hoog, G.S. (2014) Cyphellophora and its relatives in Phialophora: 475 biodiversity and possible role in human infection. Fungal Divers 65, 17–45. 476 477 Florencio, C., Couri, S. and Farinas, C.S. (2012) Correlation between agar plate screening and 478 solid-state fermentation for the prediction of cellulase production by Trichoderma strains. Enzyme 479 Res 2012, e793708. 480 481 Fujii, T., Hoshino, T., Inoue, H. and Yano, S. (2014) Taxonomic revision of the cellulose-482 degrading fungus Acremonium cellulolyticus nomen nudum to Talaromyces based on phylogenetic 483 analysis. FEMS Microbiol Lett 351, 32–41. 484 485 Garzlllo, A.M.V., Di Paolo, S., Ruzzi, M. and Buonocore, V. (1994) Hydrolytic properties of 486 extracellular cellulases from Pleurotus ostreatus. Appl Microbiol Biotechnol 42, 476–481. 487 488 Gohel, H.R., Contractor, C.N., Ghosh, S. K. and Braganza, V.J. (2014) A comparative study of 489 various staining techniques for determination of extra cellular cellulase activity on Carboxy 490 Methyl Cellulose (CMC) agar plates. Int J Curr Microbiol Appl Sci 3, 261–266. 491 492 Goukanapalle, P.K.R., Kanderi, D.K., Rajoji, G., Shanthi Kumari, B.S. and Bontha, R.R. (2020) 493 Optimization of cellulase production by a novel endophytic fungus Pestalotiopsis microspora 494 TKBRR isolated from Thalakona forest. Cellulose 27, 6299–6316. 495 496 Harnpicharnchai, P., Champreda, V., Sornlake, W. and Eurwilaichitr, L. (2009) A thermotolerant 497 β-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass 498 conversion to sugars. Protein Expr Purif 67, 61–69. 499 500 Havermans, J., Aziz, H.A. and Scholten, H. (2003) Non destructive detection of iron gall inks by 501 means of multispectral imaging part 1: Development of the detection system. Restaurator 24, 55-502 60. 503 504 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Henniges, U., Prohaska, T., Banik, G. and Potthast, A. (2006) A fluorescence labeling approach 505 to assess the deterioration state of aged papers. Cellulose 13, 421–428. 506 507 Herculano, P.N., Lima, D.M.M., Fernandes, M.J.S., Neves, R.P., Souza-Motta, C.M. and Porto, 508 A.L.F. (2011) Isolation of cellulolytic fungi from waste of castor (Ricinus communis L.). Curr 509 Microbiol 62, 1416–1422. 510 511 Itävaara, M., Siika-aho, M. and Viikari, L. (1999) Enzymatic degradation of cellulose-based 512 materials. J Polym Environ 7, 67–73. 513 514 Jaén García, L.F. (2019) José Luis Coto Conde y la transformación del Archivo Nacional de Costa 515 Rica. Revista del Archivo Nacional de Costa Rica 83, 54–72. 516 517 Jeong, M.J., Kang, K.Y., Bacher, M., Kim, H.J., Jo, B.M. and Potthast, A. (2014) Deterioration of 518 ancient cellulose paper, Hanji: evaluation of paper permanence. Cellulose, 21, 4621–4632. 519 520 Jia, Y., Yin, L., Zhang, F., Wang, M., Sun, M., Hu, C., Liu, Z., Chen, Y., Lui, J. and Pan, J. (2020) 521 Fungal community analysis and biodeterioration of waterlogged wooden lacquerware from the 522 Nanhai no. 1 shipwreck. Appl Sci 10, e3797. 523 524 Johnsen, H.R. and Krause, K. (2014) Cellulase activity screening using pure 525 carboxymethylcellulose: Application to soluble cellulolytic samples and to plant tissue prints. Int 526 J Mol Sci 15, 830–838. 527 528 Kasana, R.C., Salwan, R., Dhar, H., Dutt, S. and Gulati, A. (2008) A rapid and easy method for 529 the detection of microbial cellulases on agar plates using Gram’s iodine. Curr Microbiol 57, 503–530 507. 531 532 Kepler, R., Ban, S., Nakagiri, A., Bischoff, J., Hywel-Jones, N., Owensby, C.A. and Spatafora, 533 J.W. (2013) The phylogenetic placement of hypocrealean insect pathogens in the genus 534 Polycephalomyces: An application of One Fungus One Name. Fungal Biol 117, 611–622. 535 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ 536 Koh, S., Mizuno, M., Izuoka, Y., Fujino, N., Hamada-Sato, N. and Amano, Y. (2021) Xylanase 537 from marine filamentous fungus Pestalotiopsis sp. AN-7 was activated with diluted salt solution 538 like brackish water. J Appl Glycosci, 68, 11–18. 539 540 Ling, Z., Wang, T., Makarem, M., Cheng, H.N., Kang, X., Bacher, M., Potthast, A., Rosenau, T., 541 King, H., Delhom. C.D., Nam, S., Edwards, J.V., Kim, S.H., Xu, F. and French, A.D. (2019) 542 Effects of ball milling on the structure of cotton cellulose. Cellulose 26, 305–328 543 544 Lodhi, M.A., Ye, G.N., Weeden, N.F. and Reisch, B.I. (1994) A simple and efficient method for 545 DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12, 6-13. 546 547 Mazzoli, R., Giuffrida, M.G. and Pessione, E. (2018) Back to the past: “find the guilty bug—548 microorganisms involved in the biodeterioration of archeological and historical artifacts”. Appl 549 Microbiol Biotechnol 102, 6393–6407. 550 551 Mesquita, N., Portugal, A., Videira, S., Rodríguez-Echeverría, S., Bandeira, A.M.L., Santos, 552 M.J.A. and Freitas, H. (2009) Fungal diversity in ancient documents. A case study on the Archive 553 of the University of Coimbra. Int Biodeterior Biodegradation 63, 626-629. 554 555 Mwamburi, L.A. (2021) Endophytic fungi, Beauveria bassiana and Metarhizium anisopliae, 556 confer control of the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: 557 Noctuidae), in two tomato varieties. Egypt J Biol Pest Control 31, e7. 558 559 Negi, A. and Sarethy, I. P. (2019) Microbial biodeterioration of cultural heritage: events, 560 colonization, and analyses. Microb Ecol 78, 1014–1029. 561 562 Negulescu, I., Hyojung, K., Collier, B.J. Collier, J.R. and Pends, A. (1998) Recycling cotton from 563 cotton/ polyester fabrics. Text Chem Color 30, 31–35. 564 565 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ O'Donnell, K. and Cigelnik, E. (1997) Two divergent intragenomic rDNA ITS2 types within a 566 monophyletic lineage of the Fungus Fusarium are nonorthologous. Mol Phylogenetics Evol 7, 103-567 116. 568 569 Palla, F., Barresi, G. (2017) Biotechnology and conservation of cultural heritage. Springer, Cham. 570 Pinheiro, A.C., Sequeira, S.O. and Macedo, M.F. (2019) Fungi in archives, libraries, and museums: 571 a review on paper conservation and human health. Crit Rev Microbiol 45, 686–700. 572 573 Ponizovskaya, V.B., Rebrikova, N.L., Kachalkin, A.V., Antropova, A.B., Bilanenko, E.N. and 574 Mokeeva, V.L. (2019) Micromycetes as colonizers of mineral building materials in historic 575 monuments and museums. Fungal Biol 123, 290–306. 576 577 Posada, F. and Vega, F.E. (2006) Inoculation and colonization of coffee seedlings (Coffea arabica 578 L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). 579 Mycoscience 47, 284–289. 580 581 Povolotckaia, A.V., Pankin, D.V., Sazanova, K.V., Petrov, Y.V., Kurganov, N.S., Mikhailova, A. 582 A., Povolotckiy, A.V., Kurochkin, A.V., Vlasov, A.D., Gonobobleva, S.L., Galushkin, A.A. and 583 Hosid, E.G. (2019) Biodamage to paper by micromycetes under experimental conditions: a study 584 by vibrational spectroscopy methods. Opt Spectrosc 126, 354–359. 585 586 Proniewicz, L.M., Paluszkiewicz, C., Wesełucha-Birczyńska, A., Majcherczyk, H., Barański, A. 587 and Konieczna, A. (2001) FT-IR and FT-Raman study of hydrothermally degradated cellulose. J 588 Mol Struct 596, 163–169. 589 590 Puškárová, A., Bučková, M., Habalová, B., Kraková, L., Maková, A. and Pangallo, D. (2016) 591 Microbial communities affecting albumen photography heritage: a methodological survey. Sci Rep 592 6, e20810. 593 594 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Pyzik, A., Ciuchcinski, K., Dziurzynski, M. and Dziewit, L. (2021) The bad and the good-595 microorganisms in cultural heritage environments-an update on biodeterioration and biotreatment 596 approaches. Materials 14, 1–15. 597 598 Rambaut, A. (2010) FigTree v1.3.1. Institute of Evolutionary Biology, University of Edinburgh, 599 Edinburgh. http://tree.bio.ed.ac.uk/software/figtree/ 600 601 Ranalli, G. and Zanardini E. (2021) Biocleaning on cultural heritage: new frontiers of microbial 602 biotechnologies. J. Appl. Microbiol 131, 583-603. 603 604 Ranalli, G., Alfano, G., Belli, C., Lustrato, G., Colombini, M.P., Bonaduce, I., Zanardini, E., 605 Abbruscato, P., Cappitelli, F., Sorlini, C. (2005) Biotechnology applied to cultural heritage: 606 biorestoration of frescoes using viable bacterial cells and enzymes. J. Appl. Microbiol 98, 73-83. 607 608 Reddy, M.S., Murali, T.S., Suryanarayanan, T.S., Govinda Rajulu, M.B. and Thirunavukkarasu, 609 N. (2016) Pestalotiopsis species occur as generalist endophytes in trees of Western Ghats forests 610 of southern India. Fungal Ecol 24, 70–75. 611 612 Rodríguez-Rodríguez, C. E., Jesús García-Galán, M., Blánquez, P., Díaz-Cruz, M. S., Barceló, D., 613 Caminal, G., and Vicent, T. (2012) Continuous degradation of a mixture of sulfonamides by 614 Trametes versicolor and identification of metabolites from sulfapyridine and sulfathiazole. J 615 Hazard Mater 213–214, 347–354. 616 617 Romero, S.M., Giudicessi, S.L. and Vitale, R.G. (2021) Is the fungus Aspergillus a threat to 618 cultural heritage? J Cult Her 51, 107–124. 619 620 Sanjuan, T., Tabima, J., Restrepo, S., Læssøe, T., Spatafora, J.W. and Franco-Molano, A.E. (2014) 621 Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species 622 complex (Cordyceps sensu stricto). Mycologia 106, 260–275. 623 624 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Sequeira, S.O., De Carvalho, H.P., Mesquita, N., Portugal, A. and Macedo, M.F. (2019) Fungal 625 stains on paper: Is what you see what you get? Conservar Patrimonio 32, 18–27. 626 627 Shrestha, B., Kubátová, A., Tanaka, E., Oh, J., Yoon, D.H., Sung, J.M. and Sung, G.H. (2019) 628 Spider-pathogenic fungi within Hypocreales (Ascomycota): their current nomenclature, diversity, 629 and distribution. Mycol Prog 18, 983–1003. 630 631 Silva, A.G. (2011) The difficulty for conserving cultural heritage in tropical countries: the 632 experience of Rio de Janeiro City. Int Preserv News 54, 40–43. 633 634 Sterflinger, K. and Pinzari, F. (2012) The revenge of time: Fungal deterioration of cultural heritage 635 with particular reference to books, paper and parchment. Environ Microbiol 14, 559–566. 636 637 Šuchová, K., Puchart, V., Spodsberg, N., Mørkeberg Krogh, K. B. R. and Biely, P. (2020) A novel 638 GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-639 reducing end. Enzyme Microb Technol 134, e109484. 640 641 Trovão, J. and Portugal, A. (2021) Current knowledge on the fungal degradation abilities profiled 642 through biodeteriorative plate essays. Appl Sci 11, e4196. 643 644 Vahur, S., Teearu, A., Peets, P., Joosu, L. and Leito, I. (2016) ATR-FT-IR spectral collection of 645 conservation materials in the extended region of 4000-80 cm–1. Anal Bioanal Chem 408, 3373–646 3379. 647 648 Valášková, V. and Baldrian, P. (2006). Estimation of bound and free fractions of lignocellulose-649 degrading enzymes of wood-rotting fungi Pleurotus ostreatus, Trametes versicolor and 650 Piptoporus betulinus. Res Microbiol 157, 119–124. 651 652 Vieto, S., Escudero-Leyva, E., Avendaño, R., Rechnitzer, N., Barrantes-Madrigal, M.D., Conejo-653 Barboza, G., Herrera-Sancho, O. A., Chaverri, P. and Chavarría, M. (2022) Biodeterioration and 654 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ cellulolytic activity by fungi isolated from a nineteenth-century painting at the National Theatre 655 of Costa Rica. Fungal Biol 126, 101–112. 656 657 Watanabe, M., Inoue, H., Inoue, B., Yoshimi, M., Fujii, T. and Ishikawa, K. (2014) Xylanase 658 (GH11) from Acremonium cellulolyticus: Homologous expression and characterization. AMB 659 Express 4, 1–8. 660 661 White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal 662 ribosomal RNA genes for phylogenetics, PCR Protocols. Academic Press, Inc. 663 664 Zhao, J., Shi, P., Huang, H., Li, Z., Yuan, T., Yang, P., Luo, H., Bai, Y., Yao, B. (2012) A novel 665 thermoacidophilic and thermostable endo-β-1,4-glucanase from Phialophora sp. G5: Its 666 thermostability influenced by a distinct β-sheet and the carbohydrate-binding module. Appl 667 Microbiol Biotechnol 95, 947–955. 668 669 Zhao, J., Shi, P., Luo, H., Yang, P., Zhao, H., Bai, Y., Huang, H., Wang, H. and Yao, B. (2010) 670 An acidophilic and acid-stable β-mannanase from Phialophora sp. P13 with high mannan 671 hydrolysis activity under simulated gastric conditions. J Agric Food Chem 58, 3184–3190. 672 673 674 675 676 677 678 679 680 681 682 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Tables 683 684 Table 1. Chemical, substrate, and ink characterization of the historic documents from Costa 685 Rica. 686 Document Sections analyzed for chemical characterization Sections analyzed for fungal isolation Paper composition Ink chemical elements detected Independence Act, 1821 Pages 1-3 Pages 1-3 Cotton Fe, Ca, Zn, K Cloudy Days Act, 1821 Page 1 Page 2 Full document Cotton Cellulose acetate Fe, Ca, Zn, K, S, Cl, Pb Fe, Ca, Zn Political Constitution, 1949 (1991 reproduction) Full document Cover and Slavery Abolition page Wood cellulose Not determined Guatemalan Series 1539 Full document Pages 1,8,3,17 Cotton Not determined Guatemalan Series 1549 Full document Pages 2,5,9,10 Cotton Not determined 687 688 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Table 2. Identification (ID); BLAST results using ITS, TEF1 and TUB2; and origin of documents. Names in bold indicate the 689 assigned classification. 690 691 Isolate number Origin Closest match and accession number / % similarity * ID with ITS ID with TEF1 ID with TUB2 1539-A1P Guatemalan series, 1539 Purpureocillium lilacinum MZ359582 / 99.3 Purpureocillium lilacinum MH613753 / 100 Purpureocillium lilacinum JQ965112 / 99.8 1549-1A1P Guatemalan series, 1549 Penicillium sp. FJ752622 / 99.81 - Penicillium compactum KM973202 / 98.6 1549-4A1C Unidentified Herpotrichiellaceae KJ612089 / 90.48 no match no match AI1-A1C Independence Act, 1821 Cladosporium sp. OK274323 / 100 - Cladosporium oxysporum EF101455 / 95.5 AI3-A1P Aspergillus hiratsukae MN347034 / 100 - Aspergillus hiratsukae MH644026 / 100 CP1-A1C Political Constitution, 1949 (1991 reproduction) Periconia sp. KP128003 / 99.80 no match no match CP1-A1P Cladosporium sp. OK274323 / 92.48 - Cladosporium sp. JQ217373 / 97 CP1-A2C Pestalotiopsis microspora OK254042 / 100 - Neopestalotiopsis clavispora OM328818 / 98.2 CP1-A2P Cladosporium sp. EF504401 / 100 - - CP1-A3C Trametes hirsuta GQ280373 / 100 - - CP1-A3P Unidentified Psathyrellaceae JQ922137 / 100 - no match CP2-A1C Unidentified Pleosporales KP263091 / 92.42 - Biatriospora sp. MF588919 / 86 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ CP2-A1P Purpureocillium lilacinum MZ359582 / 99.88 - Purpureocillium lilacinum GU968702 / 100% CP2-A2C Pestalotiopsis trachycarpicola MZ453106 / 99.82 Neopestalotiopsis sp. KR493607 / 100 Pestalotiopsis kenyana KX895360 / 100 CP2-A2P Coprinellus sp. MK307658 / 99.84 - - CP2-A3C Acremonium persicinum JQ599382 / 99.42 - - CP2-A3P Beauveria aff. bassiana MZ618707 / 100 - Xenoacremonium recifei KM232105 / 89 CP2-A4C Acremonium persicinum JQ599382 / 100 - - CP2-A4P Cyphellophora aff. pluriseptata MH063042.1 / 91.7 - Cyphellophora sp. LR814116 / 80% CP2-A5C Penicillium aff. sumatraense MH864547 / 100 - - ND1-A1P Cloudy Days Act, 1821 Penicillium steckii MZ568311 / 100 - Penicillium steckii MW196656 / 100 ND2-A1P Cladosporium sp. OK242741 / 100 - Cladosporium aff. oxysporum KU216745 / 99.7 *If the percent identity is less than 80% and query coverage less than 50%, then the result is indicated as “no match.” 692 693 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ Table 3. Enzymatic index (EI) registered by fungal isolates (± indicates standard deviation based 694 on three replicates). 695 Isolate code Taxonomy Enzymatic Index (EI) CP2-A4P Cyphellophora aff. pluriseptata 4.0 ± 0.3 ND1-A1P Penicillium steckii 3.3 ± 0.3 CP1-A2P Cladosporium sp. 3.3 ± 0.1 CP2-A1P Purpureocillium lilacinum 3.2 ± 0.0 1549-4A1C Herpotrichiellaceae 3.1 ± 0.4 CP2-A2C Pestalotiopsis kenyana 3.1 ± 0.5 CP2-A3P Beauveria aff. bassiana 3.1 ± 0.3 CP1-A1P Cladosporium sp. 3.0 ± 0.1 CP2-A5C Penicillium aff. sumatraense 2.9 ± 0.7 CP2-A1C Pleosporales 2.8 ± 0.2 AI3-A1P Aspergillus hiratsukae 2.8 ± 0.0 1549-1A1P Penicillium compactum 2.6 ± 0.9 AI1-A1C Cladosporium sp. 2.6 ± 0.1 CP1-A1C Periconia sp. 2.5 ± 0.3 ND2-A1P Cladosporium aff. oxysporum 2.5 ± 0.2 CP1-A3P Psathyrellaceae 2.2 ± 0.2 CP2-A2P Coprinellus sp. 2.0 ± 0.3 CP2-A4C Acremonium persicinum 1.8 ± 0.1 CP1-A2C Neopestalotiopsis clavispora 1.7 ± 0.1 CP2-A3C Acremonium persicinum 1.6 ± 0.2 Control Pleurotus ostreatus 1.3 ± 0.1 CP1-A3C Trametes hirsuta 1.1 ± 0.0 1539-A1P Purpureocillium lilacinum 0 Figure captions Figure 1. Historic documents from Costa Rica analyzed for chemical and substrate characterization. A,B. Independence Act. C. Signs of deterioration on the Independence Act, including yellow spots around the letters. D. Cloudy Days Act. E. Humidity mark on Cloudy Days Act. F. Oxidation signs around the ink from Cloudy Days Act. G. 1949 Political Constitution .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ (1991 reproduction). H. Signs of microbiological contamination on 1949 Political Constitution (1991 reproduction). I. Yellow spots on 1949 Political Constitution (1991 reproduction). J. Fragment of the 1539 Guatemalan Series. K. Fragment of the 1549 Guatemalan Series. L,M. Signs of leakage and humidity on 1539-1549 Guatemalan Series. Figure 2. Bayesian Inference consensus cladogram based on nrDNA ITS sequences. Posterior probabilities are indicated at branches. LnL = -13564.21 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/ .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 12, 2022. ; https://doi.org/10.1101/2022.06.12.495835doi: bioRxiv preprint https://doi.org/10.1101/2022.06.12.495835 http://creativecommons.org/licenses/by-nc-nd/4.0/