Fungal and Bacterial Biodeterioration of Outdoor Canvas Paintings: The Case of the Cloisters of Quito, Ecuador
Abstract
The historic center of Quito, Ecuador, was one of the first World Cultural Heritage Sites declared by UNE-ISCO in 1978. here are numerous religious buildings built during the Spanish colonial period reflecting the cultural her- itage in this area. Between them, the cloisters of San Francisco, Santo Domingo, and Santa Clara should be highlighted. The specific problems of conservation of the outdoor canvas paintings are not well known at the moment. The objective of this paper is to achieve a conservation study of the canvas paintings exhibited in these three cloisters of the historic center of Quito in order to identify the microbial agents and the main bioclimatic parameters of deterioration. For this, a study of the state of conservation of five canvas paintings has been carried out, as well as a sampling and identification of the main microorganisms present on the obverse and reverse of the works, employing diverse techniques, traditional and biomolecular ones. An analysis of climatic conditions has also been achieved in the cloister of San Francisco. The results of the study indicate that the exhibition conditions in the cloisters are really problematic for the conservation of paintings. Important biodeteriorating agents have been isolated, including fungi and bacteria species belonging, among others, to the genera Bacillus, Penicillium, Alternaria, Mucor, and Aspergillus. We have also researched its relationship with the deterioration state of the artworks and the exhibi ion conditions in each case, proposing guidelines for the proper conservation of this important World Cultural Heritage.
Full text
Fungal and Bacterial Biodeterioration of Outdoor Canvas Paintings: The Case of the Cloisters of Quito, Ecuador Fernando Poyatos-Jiménez,a,b,* Fátima Morales,a,c,* Rosa Morales-Carrera,d Silvia Boffo,a Antonio Giordano,a & Julio Romero-Noguerab,* aSbarro Institute for Cancer Research and Molecular Medicine, Department of Biology, Temple University, Philadelphia, Pennsylvania, USA; bDepartment of Painting, Faculty of Fine Arts, University of Seville, Spain; cDepartment of Preventive Medicine and Public Health, University of Seville, Spain; dDepartment of Microbiology, School of Engineering Sciences and Industry, UTE University, Quito, Ecuador *Address all correspondence to: Fernando Poyatos-Jiménez and Julio Romero-Noguera, Dpto. Pintura, Facultad de Bellas Artes, Universidad de Sevilla, c/ Laraña 3, 41003 Sevilla, Spain; Tel.: +34954488176; Fax: +34954487763, E-mail: [email protected]; [email protected]; or Fátima Morales Marín, Dpto. Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de Sevilla, Avda. Doctor Fedriani, s/n, 41009 Sevilla, Spain; Tel.: +34954551771; Fax: +34954559827, E-mail: [email protected] ABSTRACT: 7KHKLVWRULFFHQWHURI4XLWR(FXDGRUZDVRQHRIWKH¿UVW:RUOG&XOWXUDO+HULWDJH6LWHVGHFODUHGE\81(- 6&2LQ7KHUHDUHQXPHURXVUHOLJLRXVEXLOGLQJVEXLOWGXULQJWKH6SDQLVKFRORQLDOSHULRGUHÀHFWLQJWKHFXOWXUDOKHULWDJHLQWKLVDUHD%HWZHHQWKHPWKHFORLVWHUVRI6DQ)UDQFLVFR6DQWR'RPLQJRDQG6DQWD&ODUDVKRXOGEHKLJKOLJKWHG 7KHVSHFL¿FSUREOHPVRIFRQVHUYDWLRQRIWKHRXWGRRUFDQYDVSDLQWLQJVDUHQRWZHOONQRZQDWWKHPRPHQW7KHREMHFWLYH of this paper is to achieve a conservation study of the canvas paintings exhibited in these three cloisters of the historic center of Quito in order to identify the microbial agents and the main bioclimatic parameters of deterioration. For this, a VWXG\RIWKHVWDWHRIFRQVHUYDWLRQRI¿YHFDQYDVSDLQWLQJVKDVEHHQFDUULHGRXWDVZHOODVDVDPSOLQJDQGLGHQWL¿FDWLRQ RIWKHPDLQPLFURRUJDQLVPVSUHVHQWRQWKHREYHUVHDQGUHYHUVHRIWKHZRUNVHPSOR\LQJGLYHUVHWHFKQLTXHVWUDGLWLRQDO and biomolecular ones. An analysis of climatic conditions has also been achieved in the cloister of San Francisco. The results of the study indicate that the exhibition conditions in the cloisters are really problematic for the conservation of paintings. Important biodeteriorating agents have been isolated, including fungi and bacteria species belonging, among others, to the genera Bacillus, Penicillium, Alternaria, Mucor, and Aspergillus:HKDYHDOVRUHVHDUFKHGLWVUHODWLRQVKLS ZLWKWKHGHWHULRUDWLRQVWDWHRIWKHDUWZRUNVDQGWKHH[KLELWLRQFRQGLWLRQVLQHDFKFDVHSURSRVLQJJXLGHOLQHVIRUWKHSURSHU FRQVHUYDWLRQRIWKLVLPSRUWDQW:RUOG&XOWXUDO+HULWDJH KEY WORDS: biodeterioration, canvas painting, cloisters, conservation, Quito I. INTRODUCTION The historic center of the city of Quito, Ecuador, ZDVGHFODUHG81(6&2:RUOG+HULWDJH&HQWUHLQ 1978. Numerous buildings built during the Spanish colonial period stand out, responding to urban settlements that the different religious orders were creating throughout the centuries, holding an invalu- DEOHPDWHULDOKHULWDJH2QHRIWKHSDUWLFXODULWLHVRI the ensemble is the exhibition of cultural assets in the cloisters, with important pictorial series, as well as altarpieces, mural paintings, sepulchres and stone sculptures. The cloisters are spacious and illuminated SODFHV ZKLFK KDV IUHTXHQWO\ OHG UHOLJLRXV LQVWLWX- tions to the exhibition and relocation in these spaces RIZRUNVRIDUWDQGFRPSOHWHSLFWRULDOVHULHVVXFKDV those dedicated to the Lives of the Saints (Fig. 1). 2I SDUWLFXODU LQWHUHVW DUH WKRVH EHORQJLQJ WR the convents of San Francisco, Santo Domingo DQG 6DQWD &ODUD ORFDWHG LQ WKH VRXWKHUQ DUHD RI WKH KLVWRULF FHQWHU )LJ ZKHUH ZRUNV RI JUHDW TXDOLW\GLYHUVHRULJLQDQGVWDWHRIFRQVHUYDWLRQDUH exposed.1–3 In the colonial paintings of Quito, it is GLI¿FXOWWRHVWDEOLVKKRPRJHQHRXVWHFKQLFDODUWLVWLF and stylistic features that respond to the existence of a local pictorial school, as happens with sculpture. 7KHH[LVWLQJGRFXPHQWDWLRQUHJDUGLQJWKHZRUNVLV DOVRVFDUFHDVDUHÀHFWLRQRIWKHOLWWOHFRQVLGHUDWLRQ of this pictorial creation received in the past.4,5 The diverse typologies of cultural heritage exposed in these spaces are affected by deterioration Critical ReviewsTM in Eukaryotic Gene Expression, 31(3):45–63 (2021) E\%HJHOO+RXVH,QFZZZEHJHOOKRXVHFRP 45
46 Poyatos-Jiménez et al. factors. The most important ones are environmental conditions, illuminance, human deterioration and the ones caused by biological agents.6–8 7KH FOLPDWH RI 4XLWR LV FDOOHG HTXDWRULDO RU subtropical of high lands and it is mild throughout the year . It is characterized by the high contrast between the day and night, with maximum and PLQLPXPWHPSHUDWXUHVUDQJLQJIURPWR& ZLWKDQDQQXDODYHUDJHRI&7KHUHLVDURWDWLRQ between a dry and a wet season, in which there are IUHTXHQWUHODWLYHKXPLGLW\5+SHDNVLQWKHIRUP of torrential rain . The dry season runs from June FIG. 1:&ORLVWHURI6DQ)UDQFLVFR&RQYHQW$%DQG6DQWR'RPLQJR&RQYHQW& FIG. 2: Details of the historic center of Quito. Shown is the location of the three cloisters of the research: San Fran- FLVFR$6DQWD&ODUD%DQG6DQWR'RPLQJR& Critical ReviewsTM in Eukaryotic Gene Expression
Volume 31, Issue 3, 2021 )XQJDODQG%DFWHULDO%LRGHWHULRUDWLRQRI2XWGRRU&DQYDV3DLQWLQJV 47 to September. Temperatures rise somewhat more than the rest of the year, hovering around 20 ° &RI PD[LPXPDYHUDJHWHPSHUDWXUHZLWKSHDNVWKDWFDQ rarely reach 30 ° &'XULQJWKHQLJKWVWKHWHPSHUD- tures drop sharply, with the minimum being 9 ° & 7KHZHWVHDVRQJRHVIURP2FWREHUWR0D\ZLWK mild temperatures of around 14 &PD[LPXPRI& during the day and 8°&GXULQJWKHQLJKW . Although Quito is 2850 meters above sea level (MASL), it almost never snows in the city. According to data from WKH1DWLRQDO,QVWLWXWHRI0HWHRURORJ\DQG+\GURORJ\ RI (FXDGRU ,1$0+, FROOHFWHG GXULQJ WKH \HDUV WRWKHDYHUDJHPRQWKO\PD[LPXP5+LQ each year was very high, 79%, and the minimum was 7KHPD[LPXPDEVROXWH5+UHJLVWHUHGWKURXJK- out the period was 100% and the minimum was 22%. :LWKUHJDUGWRWKHH[KLELWLRQRISLFWRULDOZRUNVRIDUW the environmental conditions described pose a double ULVNVLQFHWKH\FRPELQHKLJK5+OHYHOVZLWKODUJH GDLO\DQGVHDVRQDOÀXFWXDWLRQV3,9,10 ,Q SLFWRULDO DUWZRUN WKH ELRGHWHULRUDWLRQ SUR- cesses are often induced by insects, Microchiroptera, fungi, and bacteria.11,12 Important studies have been published, trying to determinate the microbial diversity associated to mural paintings and frescoes.13–17+RZHYHUWKHZRUNVGHGLFDWHGWRFDQYDV or wood painting are more scarce,18–27 because it be- FRPHVPRUHGLI¿FXOWWRJHWLQIRUPDWLRQDERXWWKH scope of these processes in easel paintings exhibited in outdoor environments. The main factors responsible for the growth of microorganisms in paintings are the chemical com- SRVLWLRQRIWKHDUWZRUNWKHDYDLODELOLW\RIQXWULHQWV and the environmental conditions, highlighting as HVVHQWLDOIDFWRUVWKHOHYHOVRI5+DQGZDWHUDFWLYLW\28 The effects of microbial biodeterioration in pictorial DUWZRUNFDQEHREVHUYHGLQERWKVLGHVWKHREYHUVH and reverse.29&DQYDVSDLQWLQJVSUHVHQWGLYHUVHPD- terials that can be altered by microorganisms. Apart from the lipid components of the pictorial layer, there are protein materials in its preparation layer (animal glues), cellulose in the support (canvas or wood) and generally terpenic natural varnishes in the protection layer. 7KH\FDQDOVRUHVSRQGWRPL[WHFKQLTXHVVXFK as egg tempera, that holds an additional protein component, or even containing new natural or synthetic SRO\PHULF PDWHULDOV LQ ZRUNV WKDW KDYH EHHQ UH- stored. Normally, the deterioration of the canvas oil SDLQWLQJVEHJLQVRQWKHUHYHUVHDVDFRQVHTXHQFHRI the easier biodegradation of the support, the presence of other nutrients and the existence of a micro- FOLPDWHRIKLJKHU5+EHFDXVHRIWKHFRQWDFWZLWK ZDOOVRUZHWDQGFROGVXUIDFHV2QO\XQGHUFRQGLWLRQVRIKLJK5+PDLQWDLQHGRYHUWLPHWKHELRGHWH- rioration could begin on the pictorial surface.13 Fungal growth can cause the deterioration of the pictorial layer as the hyphae penetrate through the layers of the canvas,30 a process that is aggravated (not only in fungi, but also in the case of bacterial biodeterioration patterns) by the excretion of metabolites such as organic and inorganic acids and the production of extracellular enzymes, which lead to the deterioration of materials and the separation and loss of pictorial layers, preparation layer, varnish and support, as well as discolorations and the appearance of stains.13,10,31 The main enzymatic activities involved in the deterioration of paintings caused by microorganisms are due to: i) enzymes of glucosidase activity, that affect to the support, ii) lipases, which catalyze hydrolysis of ester bonds of triacyl-glycerols at the interface between an insoluble substrate and water,32 iii) esterases, which act only on water-soluble substrates,32 iv) endo-N-acetyl-glucosaminidases acting on murein or chitin, and v) proteases. 7KH NQRZOHGJH RI WKH PLFURELRWD DQG HQYL- ronmental conditions is important for the proper FRQVHUYDWLRQRIWKHSLFWRULDODUWZRUNDVZHOODVWR choose the most appropriate suitable restoration methods.19,21,22,30,33,34 The objective of this research is to provide information about the agents of deterioration that affect this particular type of exhibited DUWZRUNFDQYDVSDLQWLQJVDQGLWVUHODWLRQVKLSZLWK the environmental conditions. For that, a follow-up KDVEHHQFDUULHGRXWWRNQRZWKHPDLQELRFOLPDWLF SDUDPHWHUVLQWKHFORLVWHUVRI4XLWR5+WHPSHUDWXUHDQGWKHLUGDLO\DQGVHDVRQDOFKDQJHV:HKDYH VHOHFWHG¿YHSLFWRULDODUWZRUNVZKLFKDUHUHSUHVHQWDWLYH RI WKH HQVHPEOH ZKHUH ZH KDYH LGHQWL¿HG fungi and bacteria by conventional and molecular ELRORJ\PHWKRGVLQRUGHUWRREWDLQD¿QJHUSULQWLQJ of the regular microbiota in this environment, and we have put it in relation with the exhibition conditions. The information obtained has been compared
48 Poyatos-Jiménez et al. with the recommendations of the international institutions for the preventive conservation of canvas paintings. General recommendations have been proposed to improve the preservation of this important typology of cultural heritage. II. MATERIALS AND METHODS A. Studied Paintings 7KHVWXGLHGDUWZRUNVUHVSRQGWRGLYHUVHDUWLVWLFPR- ments, being carried out by different artists, whose WHFKQLTXHZHFRXOGSODFHFKURQRORJLFDOO\IURPWKH second half of the seventeenth century to the end RIWKHHLJKWHHQWKFHQWXU\7KHDUWLVWLFTXDOLW\RIWKH paintings is also uneven, although the aspect that we are most interested in evaluating is their material constitution. They are painted on canvas, following WKHXVXDOZRUN SURFHVVRIWKHWLPHD SUHSDUDWLRQ base made of animal glue and inorganic materials, such as plaster and calcite, with the pictorial stratum RYHUFRPSRVHGRIOLQVHHGRLODQGSLJPHQWVDQG¿- QDOO\DSURWHFWLYHWHUSHQRLGYDUQLVK7KHDUWZRUNV are the following (Table 1): • “Appearance of the Virgin in the Porciún- FXOD´6)&RQYHQWRI6DQ)UDQFLVFR&DQ- vas painting. 205 × 271 cm. XVIII century (Anonymous). • “The Entrance of Jesus in Jerusalem” (SF2) &RQYHQWRI6DQ)UDQFLVFR&DQYDVSDLQWLQJ 310 × 208 cm. XVIII century (attributed to Bernardo Rodríguez). • ³6DQ)UDQFLVFRGH$VtV´6'&RQYHQWRI 6DQWR'RPLQJR&DQYDVSDLQWLQJ× 216 cm. XVII century (Anonymous). • ³(FVWDV\RI6DLQW7HUHVD´6'&RQYHQWRI 6DQWR'RPLQJR&DQYDVSDLQWLQJ× 212 cm. XVII century (Anonymous). • ³'LYLQH 3DVWRU´ 6& &RQYHQW RI 6DQWD &ODUD&DQYDVSDLQWLQJ× 290 cm. XVIII century (Anonymous). 2IWKHVH¿YHSDLQWLQJVIRXUDUHFXUUHQWO\H[KLELWHGLQFORLVWHUVDQGD¿IWKZDVRQO\XQWLOUHFHQWO\ This is the canvas SF1, which is currently housed in RQHRIWKHLQWHUQDOFRUULGRUVRIWKH&RQYHQWRI6DQ )UDQFLVFRZKLOHWKHZRUN³7KH(QWUDQFHRI-HVXVLQ Jerusalem” (SF2) is located in the upper north wing RIWKHPDLQFORLVWHURIWKLVEXLOGLQJ7KHZRUNVH[- hibited in Santo Domingo are exhibited in the south and west wings of the cloister, and the painting of WKH&RQYHQWRI6DQWD&ODUD³'LYLQH3DVWRU´6& is also found outside, on the access stairway to the main cloister (Fig. 3). TABLE 1:2 E YHUVHUHYHUVHDQ G VDPS OL QJDUHDVRIW K H VWXGLHGDUWZRUNV San Francisco SF1 SF2 Santo Domingo SD1 SD1 reverse SD2 SD2 reverse Santa Clara 6& 6&UHYHUVH Critical ReviewsTM in Eukaryotic Gene Expression
Volume 31, Issue 3, 2021 )XQJDODQG%DFWHULDO%LRGHWHULRUDWLRQRI2XWGRRU&DQYDV3DLQWLQJV 49 1. Historic-Artistic Characteristics and Conservation State of the Paintings ,Q RUGHU WR NQRZ WKH KLVWRU\ RI WKH DQDO\]HG DUWZRUNVDQDORJDQGGLJLWDOGDWDEDVHVZHUHFRQVXOWHG VXFKDVWKH+LVWRULFDO$UFKLYHRIWKH1DWLRQDO,QVWLWXWHRI+LVWRULFDO+HULWDJHRI(FXDGRU,13&35 and the institutional archives of religious institutions. For the study of the materials, the paintings were removed from their location and an organoleptic examination and documentation were FDUULHGRXWZLWKVWDQGDUGWHFKQLTXHVYLVLEOHSKR- tography, grazing light photography (GLP) and REVHUYDWLRQZLWKDQXOWUDYLROHWÀXRUHVFHQFHODPS (UVF). 7KH */3 VWXG\ ZDV FDUULHG RXW ZLWK REOLTXH OLJKWLQJIURPDQ:/('OLJKWVRXUFHDWDQDQJOH of 5° to 30°, which enabled surface roughness or irregularities to be seen, so that an expert eye could evaluate the paintings. To document the analysis, ZHXVHGD&DQRQGLJLWDO,;86FDPHUDZLWK a 6.2–18.6 mm focal length. Examination with ul- WUDYLROHWOLJKWZDVSHUIRUPHGZLWKD89ODPS: 9/0FRXSOHGZLWKÀXRUHVFHQWWXEHV:RI UV lighting within a range of 270–380 nm, attaining DQLOOXPLQDWHGVXUIDFHDUHDRIP:FP2). B. Monitorization of Environmental Conditions The evaluation of the environmental conditions of WKHH[KLELWHGDUWZRUNVKDVEHHQDFKLHYHGIROORZLQJWKHSURWRFROVSURSRVHGE\*LODQG(QUtTXH] 36 and Villamar.37 7KH 7HFKQRORJLFDO (TXLQRFWLDO University collaborated in this project, carrying out the study . 2QVHW +RER 8 ELW 'D- ta-Logger remote sensors were used, programmed to measure temperature ( ° &5+DQGLOOXPL- nance (lux) that were located on the north, south, east, and west walls of the courtyard of the main FORLVWHURIWKH6DQ)UDQFLVFR&RQYHQWDWDKHLJKW of 2.66 m and close to the studied paintings (Fig. 3). Measurements were carried out over a 6-month period, from mid-September 2014 to mid-March 2015, with measurement intervals every 15 minutes. The obtained data of the cloister of San Francisco is considered representative of the climatic conditions of all the studied paintings as they are held in simi- ODUFRQGLWLRQVLQWKHRWKHUWZRFORLVWHUV6DQWD&ODUD and Santo Domingo). C. Microbiological Study 1. Sampling For this study, we selected a non-invasive sam- SOLQJ WHFKQLTXH 38 based on the use of a sterile cotton swab to rub the obverse and reverse sur- IDFHV RI WKH DUWZRUNV 6SHFL¿FDOO\ ZH FROOHFWHG sample in areas of 20 cm2 with buffered peptone ZDWHU0HUFN6SDLQLQ]RQHVZKHUHVLJQVRIDO- teration compatible with biodeterioration patterns FIG. 3:/RFDWLRQRIWKHVWXGLHGDUWZRUNLQWKHFORLVWHUV and data loggers
50 Poyatos-Jiménez et al. were observed, such as changes in the colors of the painting, the appearance of stains or variations in the structure of the painted layer, both on the ob- YHUVHDQGUHYHUVHRIWKH¿YHHDVHOSDLQWLQJVXQGHU study (Fig. 4). 2. Isolation of Microorganisms All the samples collected from the easel paintings were analyzed for microorganism (bacteria and IXQJL LGHQWL¿FDWLRQ 7KH LVRODWHG EDFWHULD ZHUH VWXGLHGE\SRO\PHUDVHFKDLQUHDFWLRQ3&5,QWKH FDVHRIIXQJLWUDGLWLRQDOPRUSKRORJLFDOLGHQWL¿FD- tion was performed, except in four uncertain cases, LQZKLFKPROHFXODUWHFKQLTXHVZHUHXVHG Bacterial isolation was performed by pouring 1 mL of each sample on Petri dishes of Nutrient DJDU0HUFN6SDLQ7KLVPHGLXPSURYLGHVFDU- bon, nitrogen and mineral salts, supporting the growth of a wide range of non-fastidious bacteria that could grow on the studied paintings. Petri dishes were incubated at 37&IRUKRXUV$IWHU that, we achieved the counting and isolation of the diverse morphological colonies according to the methodology recommended by the U.S. Food and Drug Administration (2020). In the case of fungi, the isolation was carried out by pouring 1 mL of each sample into a Petri dish of Sabouraud chloramphenicol agar 0HUFN6SDLQ7KH\ZHUHLQFXEDWHGDW&IRU 15 days, after which the morphologically different colonies were counted and isolated. The iden- WL¿FDWLRQRIWKHLVRODWHGFRORQLHVZDVFDUULHGRXW E\WUDGLWLRQDODQGPROHFXODUELRORJ\WHFKQLTXHV :HHYDOXDWHG WKHNLQHWLFJURZWKRIWKH FRORQLHV at different temperatures: 20&&&DQG 35&VWDLQLQJZLWKODFWRSKHQROEOXHWRDQDO\]HWKH morphological structures with the microscope, and FRPSDULQJWKHPZLWKOLWHUDWXUHGDWDDERXWVSHFL¿F morphological characteristics .39,40 The cultures were observed daily to monitor the colony growth. After microorganism isolation, bacteria were grown in trypticase soy agar (TSA, 0HUFN 6SDLQ DQG IXQJL LQ SRWDWR GH[WURVH DJDU 3'$ 0HUFN 6SDLQ $OO PLFURRUJDQLVPV ZHUH conserved in 70% glycerol at –80& 3. DNA Extraction Genomic DNA from pure bacterial strains was extracted following the procedure described by Schabereiter-Gurtner et al. ,30 which relies on a FIG. 4:6DPSOLQJDUHDVRIWKHDUWZRUNV6'$DQG6)%& Critical ReviewsTM in Eukaryotic Gene Expression
Volume 31, Issue 3, 2021 )XQJDODQG%DFWHULDO%LRGHWHULRUDWLRQRI2XWGRRU&DQYDV3DLQWLQJV 51 chemical disruption of cells, the removal of cell wall debris, polysaccharides, and proteins by pre- FLSLWDWLRQZLWK&7$%FHW\OWULPHWK\ODPPRQLXP bromide), and the recovery of the DNA by ethanol precipitation. DNA extraction of pure fungal strains was performed according to the procedure described E\6HUWDQG6WHUÀLQJHU ,41 based on chemical and mechanical disruption of cells and the recovery RI'1$E\HWKDQROSUHFLSLWDWLRQ6SHFL¿FDOO\ZH vortex 1 cm2 of mycelium or bacteria + 500 μL of lysis buffer for 3 min and for 1 h at 65&LQ the water bath. They were vortex again for 3 min and centrifuged at 12,000 rpm for 15 min. The su- SHUQDWDQWZDVNHSWFOHDQHGZLWKWKH0LQL.LW+LJK 3XUH 3ODVPLG ,VRODWLRQ .LW 0LQL.LW 5RFKH®) following the protocol of the manufacturer. The TXDQWLW\ RI '1$ ZDV PHDVXUHG ZLWK WKH 1DQR- GURS6\VWHP7KHUPR6FLHQWL¿F® NanoDrop 2000 Spectrophotometer). 4. PCR Amplification 3&5UHDFWLRQRI'1$ZDVSHUIRUPHGLQμL volumes of a reaction mixture containing 1 μL of each primer, 2.5 ȝ/0J&O2, 0.5 ȝ/ dNTP 200 mM of each deoxyribonucleoside triphosphate, 5 ȝ/RI;3&5 buffer, 0.12 ȝ/RI7DT'1$3RO\PHUDVHȝ/ of the DNA extraction and made up to 25 ȝ/ with sterile water. Primers used for the bacterial were 341f and 907r, identifying the fragments corresponding to nucleotide positions of the Escherichia coli 16S subunit .30 For fungi, we used four combinations RIWKHXQLYHUVDOSULPHUVXVHGIRUIXQJDODPSOL¿- cation: ITS1-ITS4, ITS1-ITS2, ITS5-ITS2, and ITS3-ITS4 .42 )RU WKH DPSOL¿FDWLRQ RI IXQJDO ,76 UHJLRQV fragments of 450–600 bp in size, corresponding to the ITS1 and the ITS2 regions, and the 5.8S rRNA JHQHVLWXDWHGEHWZHHQWKHPZHUHDPSOL¿HGZLWK the primer pairs ITS1 forward and ITS4 reverse .42 :HSHUIRUPHGWKH'1$DPSOL¿FDWLRQZLWKWKH following thermocycling conditions: 10 min denaturation at 94&IROORZHGE\DPSOL¿FDWLRQ cycles of 1 min denaturation at 94°&PLQSULPHU annealing at 60&Dnd 1 min primer extension at 72&WRHQGZLWKD¿QDO extension step of 10 min at 72& 7KHJHQHWLF¿QJHUSULQWVRIWKHPLFURRUJDQLVPV ZHUHDQDO\]HGE\1HVWHG3&5XVLQJVLPLODUFRQ- ditions as before. The combinations of primers this WLPHZHUHI*&DQGUIRUEDFWHULD ,30 and ,76*&ZLWK,76IRUIXQJL .42 The total volume of each reaction was 50 μL. The mix contained 2 μL of each primer, 5 ȝ/0J&O2, 1 ȝ/ dNTP 200 mM of each deoxyribonucleoside triphosphate, 10 ȝ/ of 5×3&5EXIIHUȝ/RI7DT'1$3RO\- merase, 4 ȝ/ of the DNA extraction and made up to 50 ȝ/ with sterile water. The thermocycling conditions were: 5 min denaturation at 94& IROORZHGE\DPSOL¿FDWLRQF\Fles of 1 min denaturation at 94°&PLQSULPHUDQQHDOLQJDW& and 1 min primer extension at 72&WRHQGZLWKD ¿QDl extension step of 5 min at 72&1HVWHG3&5 products were electrophoresed in 3% agarose gel, containing ethidium bromide for 0.5 h at 100 V in 7%(EXIIHU7KHDPSOLFRQVZHUHSXUL¿HGXVLQJWKH =\PRFOHDQ*HO'1$5HFRYHU\.LW 5. Sequencing and Phylogenetic Analysis 7KHF\FOHVHTXHQFLQJUHDFWLRQVZHUHSHUIRUPHGLQ 12-ȝ/YROXPHVFRQWDLQLQJQJRI'1$ȝ0 of primer, and made up to 12 ȝ/ with sterile wa- WHU6HTXHQFLQJSULPHUVZHUHUIRUEDFWHULDDQG ,76IRUIXQJL7KHVHTXHQFLQJZDVSHUIRUPHGE\ Genewiz®&RPSDQ\6RXWK3ODLQ¿HOG1-86$ 7KHVHTXHQFHVREWDLQHGZHUHFRPSDUHGZLWKWKH GDWDEDVHRIWKH1DWLRQDO&HQWUHIRU%LRWHFKQRORJ\ Information (BLAST) (Bethesda, MD). 43 III. RESULTS AND DISCUSION A. State of Conservation Table 2 shows the technical aspects that determinate WKHFXUUHQWFRQVHUYDWLRQVWDWHRIWKHDUWZRUNV6RPH of the observed alterations are compatible with the existence of biodeterioration patterns in synergy ZLWKLQFRUUHFWOHYHOVRILOOXPLQDQFH5+DQGWHP- perature that have caused the physical-chemical alterations of binders and varnishes, as well as the ZHDNHQLQJRIFHOOXORVHWH[WLOH¿EHUV
52 Poyatos-Jiménez et al. TABLE 2:0D L QW\SHVRID O WHUDW L RQV L QW K HVWX GL H G DUWZRU N Alterations Previous restorations Reverse: Stretcher frame support Anverse: Pictorial layer varnish Deposits (dirt) Tears Deformations Previous restorations Deposits (dirt) Oxidation Cracked Loss paint Repaintings San Francisco SF1 X X X X X X X X X SF2 X X X X X X X X X X Santo Domingo SD1 X X X X X SD2 X X X X X X Santa &ODUD 6& XXXXX XX Critical ReviewsTM in Eukaryotic Gene Expression
Volume 31, Issue 3, 2021 )XQJDODQG%DFWHULDO%LRGHWHULRUDWLRQRI2XWGRRU&DQYDV3DLQWLQJV 53 1. Convent of San Francisco 7KH VWXGLHG DUWZRUNV SUHVHQW GLIIHUHQW VWDWHV RI FRQVHUYDWLRQDVDFRQVHTXHQFHRIWKHLUH[SRVXUHWR the outside and some unfortunate interventions of restorarion, which have favored the observed deterioration processes. In relation to the alterations produced by environmental agents, it is necessary to highlight the action of ultraviolet and visible radiation, which has produced the generalized dis- FRORUDWLRQRISLJPHQWVWKHODFNRIFRKHVLRQRIWKH pictorial layers due to photochemical deterioration of the binders, and the total loss of the layers of protective varnish. In the supports, we can observe a high degree of DJLQJRIWKHWH[WLOH¿EHUVZLWKHYLGHQWORVVRIÀH[L- bility and resistance. In the case of SF1, the type of ligament employed (“mantelillo”) helps to its dura- ELOLW\DVLWSURYLGHVPRUHUHVLVWHQFH:HGLGQRWJHW samples from the reverse side of SF2 because of a lining applied with synthetic materials. In general, WKHODFNRIPDLQWHQDQFHDQGSHULRGLFFOHDQLQJLVDOVR QRWHZRUWK\HVSHFLDOO\RQWKHEDFNRIWKHDUWZRUNV where dust and dirt accumulate (Fig. 5). The SF2 ZRUNLVSODFHGLQDYHU\ZLQG\FRUULGRUZKLFKJHQ- erates sudden movements in the painting and favors the deposit of fungal spores. All these factors enhance both the presence of microorganisms and their ability to alter pictorial materials, which are much PRUHHDVLO\DWWDFNHGE\IXQJLDQGEDFWHULDGXHWRWKH SUHYLRXVGHJUDGDWLRQFRQGLWLRQVRIWKHDUWZRUNV8 2. Convent of Santo Domingo 7KHGHWHULRUDWLRQSDWWHUQVREVHUYHGLQWKH&RQYHQW RI6DQ)UDQFLVFRUHFXULQWKLVFORLVWHU7KHDUWZRUNV located in the main cloister have been protected of direct sun light by a sunshade system in the south and southeast wings (Fig. 6). Despite the good intentions of these interventions, which are unfortunately WDNLQJSODFHLQPDQ\KLVWRULFDOEXLOGLQJVLQUHFHQW \HDUVWKH\FDQSRVHDVHULRXVULVNWRWKHH[SRVHG DUWZRUNV0RUHRYHUDSUHOLPLQDU\VXUYH\RIWKHHQ- vironmental parameters is essential, since they can favor other types of alterations, such as the retention of humidity by reducing ventilation, or an increase LQWHPSHUDWXUH6RWKHULVNRIDOWHUDWLRQVE\UDGLDWLRQLVFKDQJHGE\WKHULVNRIGDPDJHE\PRLVWXUH corrosion and biodeterioration. Remains of Microchiroptera and various bird droppings can be seen RQ WKH REYHUVH DQG UHYHUVH RI WKH VWXGLHG ZRUNV which can also favor the growth of microorganisms. FIG. 5:$%LUGH[FUHPHQWUHYHUVH6'&RQYHQWRI6DQWR'RPLQJR%$UDFKQLGQHVWUHYHUVH6&&RQYHQWRI 6DQWD&ODUD&%LUGGURSSLQJVLQSLFWRULDOVXUIDFH&RQYHQWRI6DQWR'RPLQJR'5HVWRIOHSLGRSWHURXVLQVHFWV UHYHUVH6'&RQYHQWRI6DQWR'RPLQJR
60 Poyatos-Jiménez et al. binders and varnishes) and discoloring the pigments of the pictorial layer. 7KHKLJKOHYHOVRI5+PDLQWDLQHGRYHUWKHWLPH allow the proliferation of fungi and bacteria. The research reveals the variety of the isolated species. The genus with more presence in the studied paintings have been Bacillus, Alternaria, and Penicillium¿YHVWUDLQVAspergillus and Fusarium (four strains), Mucor (three strains), and Cladosporium (two strains). Some of the isolated microorganisms are associated to a metabolic activity that can allow the alteration of various types of pictorial materials, belonging to the support, pictorial layer or protective varnishes. Therefore, we can suggest that these microorganisms could have been active deterioration agents, contributing to the alterations observed in the paintings. Most of the isolated microorganisms are common and respond to the patterns described in literature about microbial communities associated with exposed indoor paintings. Differential patterns have not been described at the moment due to the semitropical climate and the special exhibition FRQGLWLRQVRIWKHVWXGLHGDUWZRUNV7KHVLJQL¿FDQW presence of bacteria can be considered typical of this type of environmental conditions, since they UHTXLUHKLJKHU OHYHOV RI ZDWHU DFWLYLW\WKDQWKRVH necessary in the case of fungi. Moreover, it does not usually occur in controlled spaces, such as conventional museums. It should be mentioned that as some of the iden- WL¿FDWLRQVWXGLHVKDYHEHHQFDUULHGRXWH[FOXVLYHO\ from cultures, it is possible that there are spores of other microorganisms that have not been developed in vitro, but that can be activated under favorable conditions in the cloister environment. In indoor ambients, the biodeterioration processes are more often starting from the reverse of the paintings because of the abundance of nutrients and moisture in the VSDFHEHWZHHQWKHFDQYDVDQGWKHZDOO+RZHYHU under exposure conditions such as those described, WKH RSSRVLWH PD\ RFFXU DV D FRQVHTXHQFH RI WKH high amount of airborne microorganisms deposited RQWKHSDLQWHGVXUIDFHDQGKLJK5+HQYLURQPHQWV stable over the time. For all these reasons, it is essential to establish a preventive conservation program to ensure the VWDELOLW\ RI WKH H[KLELW DUWZRUNV RQ GLVSOD\ 7KLV program should include a daily control of environmental conditions and maintenance of the paintings, LQFOXGLQJDSHULRGLFVXSHU¿FLDOFOHDQLQJRIWKHRE- verse, reverse and protection frames, as well as a VRXQGLQJ RI WKH FDSLOODULW\ RU ¿OWUDWLRQ KXPLGLW\ OLQNHGWRWKHZDOOV7RDYRLGVXSHU¿FLDOGHSRVLWVLW is also important to properly tension the canvases to the frames, as well as to control the stability of WKHZRRGVWKDWPDNHWKHPXS5HVWRUDWLRQZRUNVDUH highly recommended in the paintings belonging to WKH&RQYHQWRI6DQ)UDQFLVFR$OWKRXJKWKH\KDYH been restored in the past, their conservation state DQGWKHZLFNHGHYROXWLRQRIWKHWUHDWPHQWVGHPDQG urgent improvements. 5HJDUGLQJWKH5+DQGWHPSHUDWXUHFRQGLWLRQV their control is almost impossible in current conditions, due to the open exhibition spaces and the large IRUPDWRIWKHSDLQWLQJV$VD¿UVWSUHYHQWLYHVWHSLW is essential to control these parameters by data logger systems with capacity of continuos storage to GHWHFWOHYHOVRI5+KLJKHUWKDQDQGÀXFWXD- tions higher than 25%.71 The possible solutions to the serious environmental problems described are of JUHDWVLJQL¿FDQFHVXFKDVWKHJOD]LQJRIWKHFORLV- ters, always avoiding the use of urns or systems that FRPSOHWHO\LVRODWHWKHZRUNVIURPWKHVXUURXQGLQJ HQYLURQPHQW DQG WKH LQVWDOODWLRQ RI +9$& FRQ- ditioning systems if it is possible due to the maximum protection gave to these cloisters. Relocation RIZRUNVLQGRRUVFRXOGDOVREHFRQVLGHUHGDWOHDVW during the wet season. The search for solutions to avoid the incidence of light radiation could be more complicated. In this VHQVHLWLVUHFRPPHQGHGWR¿QGVSDFHVZLWKQRGL- rect light exposure and not excessively windy, to DYRLGORRVHQLQJRIWKHFDQYDVDQGVXSHU¿FLDOHUR- sions. Ideally, if the cloisters were glazed, the instal- ODWLRQRI89¿OWHUVFRXOGEHVWXGLHG:HVKRXOGWDNH LQWRDFFRXQWWKHTXDOLW\TXDQWLW\DQGWLPHRIH[SRVXUHRIWKHZRUNVWRQDWXUDOOLJKWUDGLDWLRQZKLFK causes cumulative and irreversible damage to the paintings. Also, specialized training for employees in FRQWUROOLQJWKHVHSDUDPHWHUVDQGLQKDQGOLQJZRUNV is essential in order to apply the recommended procedures as effectively as possible and extend Critical ReviewsTM in Eukaryotic Gene Expression
Volume 31, Issue 3, 2021 )XQJDODQG%DFWHULDO%LRGHWHULRUDWLRQRI2XWGRRU&DQYDV3DLQWLQJV 61 the preventive biodeterioration care of the studied DUWZRUNVWRDOOWKHFORLVWHUVRIWKH0RQDVWHULHVRI Quito. ACKNOWLEDGMENT The research presented here was supported by the 6(1(6&<73URPHWHR3URMHFW6HFUHWDU\RI+LJKHU Education, Science, Technology and Innovation, Ecuador. REFERENCES &HQWURGHO3DWULPRQLR0XQGLDOGHOD 81(6&2>KRPHSDJH RQ WKH ,QWHUQHW@ ([SHGLHQWH GH &DQGLGDWXUD GH OD &LXGDGGH4XLWRDOD/LVWDGHO3DWULPRQLR0XQGLDO >FLWHG$XJ@$YDLODEOHIURPKWWSZKFXQHVFR org/en/list/2. 2. Zambrano I. La cuestión de la conservación del patrimonio mueble de San Francisco. Anales de la Universidad &HQWUDOGH(FXDGRU± 3. Poyatos-Jiménez F, Benítez J, Morales F, Giordano, A. Estudio y análisis de los procesos de alteración microbiológica en pintura sobre lienzo: Aplicaciones al patrimo- QLRTXLWHxR4XLWR(FXDGRU: U1(6&2 0HUFp-6LOYD03ODQGLUHFWRUGHUHVWDXUDFLyQ&RQYHQWR e iglesia de San Francisco. Volumen II y III. Quito, Ecuadaor: IQVWLWXWR1DFLRQDOGH3DWULPRQLR&XOWXUDO 6DQGRYDO5Xt]$3ODQGH*HVWLyQSDUDHO&RQMXQWR&RQ- ventual de San Francisco de Quito. Anales de la Universi- GDG&HQWUDOGH(FXDGRU $OOVRSS'6HDO.*D\ODUGH&,QWURGXFWLRQWRELRGHWHULRUDWLRQ&DPEULGJH&ambridge University Press; 2004. .RHVWOHU 5- .RHVWOHU 9+ &KDUROD $( 1LHWR)HUQDQ- dez FE. Art, biology, and conservation: Biodeterioration RIZRUNVRIDUW1HZ+DYHQ&7<ale University Press; 2004. 0LFKDOVN\ 6 7KH$%& PHWKRG: A ULVN PDQDJHPHQW DSSURDFKWRWKHSUHVHUYDWLRQRIFXOWXUDOKHULWDJH2W- WRZD&DQDGD&DQDGLDQ&RQVHUYDWLRQ,QVWLWXWH&&, 2016. %DUED0HUL]DOGH&(&RQVHUYDFLyQSUHYHQWLYDHQORVPX- seos de la ciudad de Quito: Diagnóstico y propuesta de un plan mínimo. Quito, Ecuador: Universidad Tecnológica (TXLQRFFLDO 2004. 3R\DWRV-LPpQH])0RUDOHV5&HYDOORV/)UHLOH&0R- rales F. Estudio y análisis de los procesos de alteración por agentes microbiológicos en obras pictóricas sobre lienzo expuestas en claustros de conventos y monasterios del centro histórico de Quito, Ecuador. Madrid, Spain: Min- LVWHULRGH(GXFDFLyQ\&XOWXUD &DQHYD * 1XJDUL 03 6DOYDGRUL 2 3ODQW %LRORJ\ IRU FXOWXUDO+HULWDJH%LRGHWHULRUDWLRQDQG&RQVHUYDWLRQ/RV ÈQJHOHV&$: GHWW\&RQVHUYDWLRQ,QVWLWXWH &LIHUUL27LDQR 30DVWURPHL * 2I PLFUREHV DQG DUW: The role of microbial communities in the degradation and protection of cultural heritage. Florence, Italy: Springer Science & Business Media; 2000. &LIHUUL20LFURELDOGHJUDGDWLRQRISDLQWLQJV$SSO(QYL- ron Microb. 1999;65:877–85. *RUEXVKLQD $$ +H\UPDQ - 'RUQLHGHQD 7 *RQ]D- OH]'HOYDOOH0.UXPEHLQ:(/DL]/3HWHUVHQ.6DL] -LPHQH]&6ZLQJV-%DFWHULDODQGIXQJDOGLYHUVLW\DQG biodeterioration problems in mural painting environments RI6W0DUWLQVFKXUFK*UHHQH.UHLHQVHQ*HUPDQ\,QW Biodeter Biodegr. 2004;53:13–24. 15. Poyatos-Jiménez F. Procesos de biodeterioro en pinturas sobre lienzo del Museo de Bellas Artes de Granada: Ex- DPHQ YLVXDO \ JUi¿FR *UDQDGD 6SDLQ: Universidad de Granada; 2007. 8U]L&GH/HR)(YDOXDWLRQRIWKHHI¿FLHQF\RIZDWHUUH- pellent and biocide compounds against microbial colonization of mortars. Int Biodeter Biodegr. 2007;60(1):25–34. 6FKHHUHU62UWHJD0RUDOHV2*D\ODUGH&0LFURELDOGH- terioration of stone monuments an updated overview. Adv Appl Microbiol. 2009;66:97–139. 'RPpQHFK&DUEy 07 2VHWH&RUWLQD / GH OD &UX] &DxL]DUHV - %ROtYDU*DOLDQR ) 5RPHUR1RJXHUD - Fernández-Vivas MA, Martín-Sánchez I. Study of the microbiodegradation of terpenoid resin-based varnishes from easel painting using pyrolysis–gas, chromatography–mass spectrometry and gas chromatography–mass VSHFWURPHWU\$QDO%LRDQDO&KHP± &DSRGLFDVD 6 )HGLD 6 3RUFHOOLD$0 =DQQRQ ' 7KH microbial community dwelling on a biodeteriorated 16th century painting. Int Biodeter Biodegr. 2010;64:722–33. 9XNRMHYLü - /MDOMHYLü *UELü 0 0RXOGV RQ SDLQWLQJV LQ 6HUELDQ ¿QH DUW PXVHXPV$IULFDQ - 0LFURELRO 5HV 2010;4(13):1453–6. /ySH]0LUDV00 0DUWtQ,<HEUD$5RPHUR1RJXHUD -%ROtYDU)(WWHQDXHU-6WHUÀLQJHU.3txDU*&RQWUL- bution of the microbial communities detected on an oil SDLQWLQJ RQ FDQYDV WR LWV ELRGHWHULRUDWLRQ 3/R6 2QH 2013;8(11):e80198. 22. López-Miras MM, Piñar G, Romero-Noguera J, Bolívar )&(WWHQDXHU-6WHUÀLQJHU.0DUWtQ,0LFURELDOFRP- munities adhering to the obverse and reverse sides of an oil painting on canvas: IGHQWL¿FDWLRQDQGHYDOXDWLRQRIWKHLU biodegradative potential. Aerobiología. 2013;29:301–14. 6WHUÀLQJHU.3LxDU*0LFURELDOGHWHULRUDWLRQRIFXOWXUDO KHULWDJHDQGZRUNVRIDUW7LOWLQJDWZLQGPLOOV"$SSO0L- crobiol Biotechnol. 2013;97(22):9637–46. 5RPHUR1RJXHUD - %ROtYDU*DOLDQR )& 5DPRV/ySH] JM, Fernández-Vivas MA, Martín-Sánchez I. Study of biodeterioration of diterpenic varnishes used in art painting&olophony and Venetian turpentine. Int Biodeter Biodegr. 2008;62(4):427–33.
62 Poyatos-Jiménez et al. 5DPRV/ySH]-0%ROtYDU*DOLDQR)&%LRGHWHULRUDWLRQ patterns found in dammar resin used as art material. Electron J Biotechnol. 2010;13(3):6–7. 26. Romero-Noguera J, López-Miras MM, Martín-Sánchez ,5DPRV/ySH]-0%ROtYDU*DOLDQR)&$QDSSURDFK to the study of the fungal deterioration of a classical art material: Mastic varnish. Electron J Biotechn. 2010;13(6):1–2. 5RPHUR1RJXHUD - 'RPpQHFK&DUEy 07 2VHWH&RUWLQD/0DUWtQ6iQFKH],%ROtYDU*DOLDQR)&$QDO\WLFDO characterisation of the biodeterioration of diterpenoid lab- GDQLFYDUQLVKHVXVHGLQSLFWRULDOWHFKQLTXHV: Sandarac and Manila copal. Int Biodeter Biodegr. 2014;90:99–105. 6iL]-LPpQH]&'HSRVLWLRQRIDLUERUQHRUJDQLFSROOXWDQWV on historic buildings. Atmos Environ. 1993;27:77–85. 6WU]HOF]\N$%3DLQWLQJVDQGVFXOSWXUHV,Q: Microbial deterioration. London: Rose Academic Press; 1981. 6FKDEHUHLWHU*XUWQHU&3LxDU*/XELW]:5|OOHNH6$Q advanced strategy to identify bacterial communities on art objects. J Microbiol Meth. 2001;45:77–87. 3R\DWRV)0RUDOHV)1LFKROVRQ$:*LRUGDQR$3K\V- LRORJ\ RI ELRGHWHULRUDWLRQ RQ FDQYDV SDLQWLQJV - &HOO Physiol. 2018;233(4):2741–51. 6ROLPDQ 1$ .QROO 0$EGHO)DWWDK <5 6FKPLG 5' Langeb S. Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermole- RYRUDQV<1LVRODWHGIURPGHVHUWVRLOLQ (J\SW3URFHVV Biochem. 2007;42(7):1090–1100. $ODNRPL +/ 3DDQDQHQ $ 6XLKNR 0/ +HODQGHU ,0 6DDUHOD 0 :HDNHQLQJ HIIHFW RI FHOO SHUPHDELOL]HUV RQ Gram negative bacteria causing biodeterioration. Appl Environ Microbiol. 2006;72:469–73. 6XLKNR0/$ODNRPL+/*RUEXVKLQD$)RUWXQH,0DU- TXDUGW-6DDUHOD0&KDUDFWHUL]DWLRQRIDHURELFEDFWHULDO and fungal microbiota on surfaces of historic Scottish monuments. 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Utrecht, the Netherlands: &entraalbureau voor Schimmelcultures; 2004. &DUULOOR/&ODYHVSDUDODLGHQWL¿FDFLyQGHORVKRQJRVGH los alimentos y forrajes. Salta, Argentina: Universidad Nacional de Salta; 2011. 6HUW % 6WHUÀLQJHU .$$ QHZ &RQLRVSRULXP VSHFLHV from historical marble monuments. Mycol Progress. 2009;9:353–9. :KLWH7$%UXQV7/HH67D\ORU-$PSOL¿FDWLRQDQGGLUHFWVHTXHQFLQJRIIXQJDOULERVRPDO51$JHQHVIRUSK\- ORJHQHWLFV,Q3&5SURWRFROV&DPEULGJH0$: Academic Press; 1990. p. 315–22. %/$67>GDWDEDVHRQWKH,QWHUQHW@%DVLF/RFDO$OLJQPHQW 6HDUFK7RRO%/$67%HWKHVGD0'1DWLRQDO&HQWHUIRU %LRWHFKQRORJ\,QIRUPDWLRQ>FLWHG6HS@$YDLODEOH from: http://www.ncbi.nlm.nih.gov/BLAST/. $6+5$( +HDWLQJ YHQWLODWLQJ DQG DLUFRQGLWLRQLQJ applications. Museums, libraries and archives. Atlanta, *$ $PHULFDQ 6RFLHW\ RI +HDWLQJ 5HIULJHUDWLQJ DQG $LU&RQGLWLRQLQJ(QJLQHHUV $6+5$( +HDWLQJ YHQWLODWLQJ DQG DLUFRQGLWLRQLQJ V\VWHPVDQGHTXLSPHQW$6+5$(+DQGERRN$WODQWD *$ $PHULFDQ 6RFLHW\ RI +HDWLQJ 5HIULJHUDWLQJ DQG $LU&RQGLWLRQLQJ(QJLQHHUV 7KRPSVRQ*0XVHXPHQYLURQPHQWQGHG2[IRUG %XWWHUZRUWK+HLQHPDQQ 6HULHV LQ &RQVHUYDWLRQ DQG Museology; 1994. 3HGHUVROL-/$QWRPDUFKL&0LFKDOVNL6$JXLGHWRULVN management of cultural heritage. Rome: International &HQWUHIRUWKH6WXG\RIWKH3UHVHUYDWLRQDQG5HVWRUDWLRQ RI&XOWXUDOSURSHUW\,&&520 48. Saltos E. Incidencia de los factores ambientales en las obras atribuidas a Miguel de Santiago ubicadas en el claustro del convento San Agustín. Quito, Ecuador: Uni- YHUVLGDG7HFQROyJLFD(TXLQRFFLDO ; 2014. 49. Boylan PJ. Running a museum: ASUDFWLFDOKDQGERRN >PRQRJUDSKRQWKH,QWHUQHW@&DQDGLDQ&RQVHUYDWLRQ,Q- VWLWXWH>FLWHG2FW@$YDLODEOHIURPKWWSV www.canada.ca/en/conservation-institute.html . +HUUiH]-$(QUtTXH]GH6DODPDQFD* 3DVWRU$UHQDV MJ, Gil Muñoz T. Manual de seguimiento y análisis de condiciones ambientales. Madrid, Spain: Ministerio de (GXFDFLyQ&XOWXUD\'HSRUWH *U]\ZDF]&00RQLWRULQJIRUJDVHRXVSROOXWDQVLQPXVH- ums environments: Tools for conservation. Los Angeles, &$: TKH*HWW\&RQVHUYDWLRQ,QVWLWXWH 6DQWRV$&HUUDGD$*DUFtD66DQ$QGUpV0$EUXVFL& 0DUTXLQD'$SSOLFDWLRQRIPROHFXODUWHFKQLTXHVWRWKH elucidation of the microbial community structure of an- WLTXHSDLQWLQJV0LFURE(FRO± 2NSDODQR]LH 2( $GHEXVR\H 6$ 7URLDQR ) 3ROR $ &DSSLWHOOL ) ,ORUL 02 (YDOXDWLQJ WKH PLFURELRORJLFDOULVNWRDFRQWHPSRUDU\1LJHULDQSDLQWLQJ: Molecular and biodegradative studies. Int Biodeter Biodegr. 2016;114:184–92. Critical ReviewsTM in Eukaryotic Gene Expression
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