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International Biodeterioration & Biodegradation 75 (2012) 83e88

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Fungal biodeterioration of historical library materials stored in Compactusmovable shelves

Matteo Montanari a,*, Valeria Melloni a, Flavia Pinzari b, Gloria Innocenti a

aDepartment of Agricoltural Sciences, Alma Mater Studiorum, Università degli Studi di Bologna, viale Fanin 46, 40127 Bologna, Italyb Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Ministero per i Beni e le Attività Culturali, via Milano 76, 00184 Roma, Italy

a r t i c l e i n f o

Article history:Received 5 January 2012Received in revised form7 March 2012Accepted 8 March 2012Available online

Keywords:Eurotium halophilicumAspergillus halophilicusBook deteriorationCultural heritageMold contaminationXerophilic fungusCompactus shelving

* Corresponding author. Tel.: þ39 (0) 512096581; fE-mail addresses: montanari.matteo@gmail.com (M

beniculturali.it (F. Pinzari).

0964-8305/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.03.011

a b s t r a c t

Mold deterioration of historical library materials in archives and libraries is a frequent and complexphenomenon that may have important economic and cultural consequences. Compactus shelving is oneof the systems utilised for the conservation of library materials, because it allows for a more efficient useof space and protection against dust deposition. However, in the last ten years there have been manyreports on single species mould infections within Compactus shelves in spite of conventional control ofenvironmental temperature and humidity to recommended standards. Contamination was commonlycharacterized by white spots of mycelium, measuring 0.5e1.0 cm in diameter and observed on volumebinding, especially those of leather, parchment or textile. Until now, the identification of the causal agentat species level has not been reported since attempts to grow it on media for subsequent identificationwere unsuccessful. Using a range of sampling techniques, including adhesive tape and nitrocellulosemembrane, and a combination of conventional culturing methods, direct microscopic observations andmolecular methods, we have for the first time identified to species level the fungus causing infectionsinside Compactus shelves.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Storage of books and documents inside structures intended fortheir preservation has created new manmade environments formicrobial species such as fungi and bacteria to inhabit (Kowalik,1980; Zyska, 1997; Nittérus, 2000). High-density storage systemsreferred to as “movable shelving” or “Compactus type shelving” areemployed by many libraries, archives and conservation institutionssuffering from limited space. These systems minimize the amountof space required for storage by compacting blocks of shelves (orcabinets of drawers) tightly together. These blocks slide alongtracks and can be moved apart (opened) for the retrieval of itemspositioned on a particular block and then moved back together(closed). Compactus shelves can also protect against dust deposi-tion (Gallo and Regni, 1998) and fire. These characteristics areadvantageous for the preservation and storage of materials but canalso be problematical when used in conservation environmentslacking efficient climate control systems. Resistance to heat and

ax: þ39 (0) 512096565.. Montanari), flavia.pinzari@

All rights reserved.

humidity exchange between the micro-environment within theCompactus units and the outer environment, can present risk tostored objects, especially when these objects are composed ofhygroscopic materials. International Federation of Library Associ-ations (IFLA) recommends 18e20 �C air temperature and 50e60%relative humidity (RH) for effective preservation of documentslike books and periodicals in archives and libraries. In practice, it isdifficult to maintain a stable temperature and relative humiditylevel, even with the benefit of air conditioning.

In the last decade, several investigations in libraries and archivesespecially within Compactus shelving blocks have reported moldcontamination and growth on volume bindings made of leather,parchment or cotton fibres. Surprisingly these reports consistentlyobserved white and irregular spots of fungal spread mainly on theexposed part of the volumes stored mainly in the lower shelves ofthe blocks. Optical and scanning electron microscopy examinationof samples collected with transparent adhesive tapes revealedthe presence of conidiophores typical of the genus Aspergillus.However, due to cultivation challenges and peculiarities ofmorphology, identification to species level was not accomplished(Pinzari and Montanari, 2011). In this study, specific identificationof this common, putative Aspergillus isolate contaminating booksstored inside Compactus shelves is reported.

M. Montanari et al. / International Biodeterioration & Biodegradation 75 (2012) 83e8884

2. Material and methods

2.1. Case study

Our investigation was accomplished in a deposit of an historicallibrary in Rome, containing books and papers stored in Compactustype shelves and contaminated by molds. Mold contamination wasspread all over the depository, mainly in the lower shelves of theCompactus blocks, in a large number of volume binding made byfabric or leather, especially on the exposed part of the book such asspine, upper edge and fore edge. Infestation pattern on the bookswas similar to those detected during previous surveys in otherarchives and libraries in Italy, consisting of irregular white, mycelialspots of variable diameter (Fig. 1). Observations of these spots atlow magnification with a digital microscope (DinoLite pro,AM413TFVW-A, Italy) demonstrated many conidiophores scatteredon the mycelium.

2.2. Sampling

Sampling of fungal elements was performed on contiguousfungal spots of eight contaminated volumes using the followingmethods: (i) sterile cotton swabs (Cultiplast e LP Italiana SPA, Italy)were wiped across fungal spots then transferred to the laboratoryin sterile tubes and used for fungal culturing and identification; (ii)pieces (6 � 2 cm) of a removable transparent adhesive tapespecifically intended for microbiological sampling (Fungi Tape;Scientific Device Lab., Glenview, Illinois, USA; 1 mm thick, n. 745),were gently pressed over spots, to collect mycelium, fruitingstructures and spores. Tapes were aseptically mounted over sterileglass slides and transferred to the laboratory. Each tape was thenbisected, one half was used for optical and electron microscopeexamination, and the other half for direct DNA extraction; (iii)sterile membranes of nitrocellulose (0.45 mm pore-size, Millipore;47 mm in diameter) where gently pressed for 10 s over mycelialspots, then immediately transferred to the surface of 6 cm Petridishes containing dichloran 18% glycerol (DG18) agar (Samsonet al., 2002).

All samples were transferred to the laboratory the same day ofcollection and immediately processed.

2.3. Agar cultures

Each swab of fungal growth was immersed in a sterile glass vialcontaining 5 ml of Ringer’s solution, and homogenized in

Fig. 1. Fungal elements on volume binding.

a sonication bath at 40� 2 kHz frequency for 5 min (Trampuz et al.,2007). Aliquots (100 ml) of homogenate were spread on each 9 cmPetri dishes (4 plates per sample) containing separately MaltExtract Agar 2% (MEA 2%), Czapek Yeast Agar with 20% sucrose(CY20S) (Pitt and Hocking, 1985) and DG18 agar. Plates wereincubated for 7e14 days at 20 �C in the dark and growth transferredon DG18 agar for isolation in pure culture.

Nitrocellulose membrane on DG18 plates were incubated at20 �C in the dark. After fungal colony appearance, membranes wereremoved and portions of mycelium were picked up for isolation inpure culture on DG18 agar plates.

Fungal isolates were identified to genus level using biometricand microscopic features (Ellis, 1971, 1976; Von Arx, 1981; Domshand Gams, 1993). Dominant fungal isolates were identified tospecies level by DNA analysis.

2.4. Optical and scanning electron microscopic observations

Two fragments (1.5 � 2 cm) were bisected from half of eachfungi tape bearing captured fungal elements. The first fragmentwas further divided in two parts (1 � 1.5 cm): one part wastransferred on a glass slide with a drop of cotton blue stain, theother part on a glass slide with a drop of fluorescein diacetate (FDA)solution (20 mg of FDA in 1 ml of phosphate buffer pH 7.3). Cottonblue staining was used for observations of morphological fungalstructures at white light microscope. FDA staining was used forobservations of active structures using an inverted epifluorescentmicroscope (Nikon eclipse T2000) equipped with a FITC filter (blueexcitation wave length: 495 nm). Active structures (positive stain-ing) were assessed by the presence of a greenish fluorescenceemanating from the cytoplasm of spores and hyphae, due to theliberation of fluorescein by enzymatic (hydrolytic) cleavage.Samples stainedwith FDAwere observed after 20min of incubationin the dark at 20 �C. All slides were examined at 400 and 600xmagnification. Micrographs were acquired using a digital cameraconnected to a PC equipped with NIS Elements software (Nikon).

The second fragment was observed using an EVO 50 ScanningElectron Microscope produced by the Carl-Zeiss Electron Micros-copy Group (Oxford, UK). Tape fragments measuring 5e10 mm indiameter were cut and mounted on to a 12 mm metal stub usingdouble-sided carbon adhesive tape. Parts of agar cultures were alsoobserved by SEM imaging. Samples were examined using a 20 kVelectron beam, both at variable pressure (VPSE) and after metalli-zation with high vacuum mode with gold (Goldstein et al., 2003).Fungal samples supported on adhesive tape were directly metal-ized without previousfixation. Elements were observed to be dry atthe time of the sampling, and fixation would have added artefacts.For observation with the scanning electron microscope (SEM),ascomata at various developmental stages on agar media wereexcised, placed in phosphate buffer (pH 7.0), and fixed in glutaral-dehyde buffer for 2 h, rinsed in distilled water and post-fixed in 2%OsO4 for 12 h at 5 �C, dehydrated in an ethanol series, taken to amylacetate, and critical point dried in a Polaron E-3000 dryer (QuorumTechnologies, Ringmer, UK) using carbon dioxide. Dried sampleswere coated at 40 mA to obtain a 15 nm-thick layer of gold (BaltecSputter Coater) and examined using the SEM EVO 50 (Carl Zeiss,Cambridge, UK).

2.5. Molecular analysis

2.5.1. DNA analysis of isolated fungal culturesDNA was extracted from mycelium of fungal isolates using the

NucleoSpin� Plant II (MACHEREY-NAGEL, Düren, Germany) forfungi, following the manufacturer instructions. ITS region of ribo-somal DNA was amplified by Polymerase Chain Reaction (PCR)

Fig. 2. White fungal colonies growing on DG18 agar plate (6 cm diameter) aftersampling with sterile nitrocellulose membrane.

M. Montanari et al. / International Biodeterioration & Biodegradation 75 (2012) 83e88 85

using for each reaction 25 ml of PCR master mix (Promega, Man-nheim, Germany), 50 pmole of both ITS1f and ITS4 as fungalprimers (White et al., 1990; Gardes and Bruns, 1993), 1 ml of BovineSerum Albumin (BSA 20 mg/ml, Fermentas), 4 ml of DNA templateand water nuclease-free to a final volume of 50 ml. The thermocy-cling programwas as follows: 3 min denaturation at 94 �C, followedby 35 cycles of 30 s denaturation at 94 �C, 30 s annealing at 55 �C,and 1 min extension at 72 �C. Ten minutes at 72 �C were used asa final extension step. Sequencing of amplified ITS regions wasperformed at Macrogen Inc. (Korea). Taxonomic identificationswere performed comparing retrieved sequences with those avail-able in the online databases provided by the National Centre forBiotechnology Information (NCBI) using the BLAST search program(Altschul et al., 1997).

2.5.2. Total DNA extraction from adhesive tapesEnvironmental DNA was extracted directly from three fungal

tape fragments (2 � 3 cm) following the protocol described bySchabereiter-Gurtner et al. (2001), modified by using a different kitfor the DNA cleaning step. This analysis was performed to comparesequences obtained using a culture-based method with those

Fig. 3. Pure cultures growing in DG18 agar plat

obtained using a culture-independent method. DNA extractioncombines enzymatic (lysozyme and proteinase K) and mechanicalsteps (freeze and thaw cycles) in the presence of cetyl-trimethylammonium bromide (CTAB) with the final step withchloroform e isoamyl alcohol (24:1). DNA in the supernatant iscleaned with the NucleoSpin� Plant II kit for soil, starting from step3 of the manufacturer’s protocol. The final elution step wasrepeated two times with 25 ml of 70 �C preheated PE Buffer. CleanedDNA extract was used for PCR amplification analysis for fungi, usingthe same protocol used for DNA from fungal culture.

2.5.3. Creation of clone libraries and sequence analysisPCR amplification from environmental DNA may produce

a mixture of different amplicons, which should be separate toaccomplish a detailed phylogenetic analysis on members of thefungal community. To separate single ITS amplified fragments,a clone library containing the ITS fungal regions was realized. Foreach DNA crude extract, PCR product was purified using Nucle-oSpin� Extract II kit Protocol (MACHEREY-NAGEL, Düren,Germany). Purified PCR products (3 ml) were cloned using pGEM-TEasy Vector System I Kit (Promega), following the manufacturerprotocol. a total of twenty white colonies positive for therecombinant plasmids were randomly selected from the threesamples (DNA crude extracts) and then subjected to a denaturationstep (6 min at 97 �C) and PCR amplification using the same primers(ITS1f and ITS4) and protocol used above. Electrophoresis of PCRproducts was conducted in agarose (1.5%) gels. Products givingpositive bands were subjected to sequencing at Macrogen Inc.Comparative sequences analysis was performed as describedabove.

3. Results

3.1. Cultivation

Sampling procedure using sterile swabs produced on agarmedia only few colonies typical of Cladosporium and Penicilliumspp. Direct plating nitrocellulose membrane on DG18 plates,produced after 6e7 days, slow-growing white colonies, slightlydepressed in the middle (Fig. 2). All colonies were of similarmorphological and biometric features. After re-isolation on DG18agar, white colonies were observed to grow at a rate of 4e7mm perweek (Fig. 3). As agar medium get drier (about 2e3 weeks afterinoculation), colonies developed cleistothecia that were globose,

e (6 cm Ø): front (left) and reverse (right).

Fig. 5. Optical micrograph on adhesive tape: conidiophore and conidia stained withcotton blue (bar ¼ 10 mm).

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white to cream in colour, 150e200 mm in diameter (Fig. 4). Theconidial state was not observed in these cultures.

3.2. Optical and SEM microscopic observations

Optical microscopic observations of adhesive tape samplesrevealed the presence of fungal structures, conidiophores andconidia, typical of Aspergillus spp. (Fig. 5). Observations of fungalelements trapped on the adhesive tape showed only conidial states.Fungal structures stained with FDA, observed with an epifluor-escent microscope using blue filter, fluoresced green revealing anenzymatic activity (Fig. 6).

All samples collected directly from books using the adhesive-tape technique examined under Scanning Electron Microscopy,showed fungal elements typical of Aspergillus species, specificallylarge conidia single or in chain, slightly ovate, echinulate withprominent scars, and conidiophores with narrow vesicles finelycovered with a layer of hairy structures (Fig. 7).

Fungal growth from agar cultures on DG18 plates, observedunder SEM, included many ascomata with asci and mature asco-spores. Hypertrophic hyphae, slightly covered with bare and shorthairs (Fig. 8), were also observed in most of the cultures. Ascomataappeared spherical to subspherical, mostly 100e150 mm in diam-eter, asci measured 10e15 mm in diam. and are spherical or nearlyso. Ascospores by SEM appeared lenticular, with rough surface andsharp furrow bordered by ridges, mostly 5 � 7 mm. The ascomataappeared characterised by a smooth surface with semi-globoseprominent structures, which make the whole structure resem-bling a “morula” (Fig. 9).

3.3. Molecular analysis

ITS sequences derived from three randomly-selected coloniesdeveloped on DG18 agar after sampling with nitrocellulosemembrane were identical and were best match by BLAST withEurotium halophilicum (An. Aspergillus halophilicus) (GenBank ID:[EF652088]), at 100% of similarity. One of them (isolate MM373)was deposited at the NCBI database (Table 1).

From the twenty selected clones of the clone library derivedfrom environmental DNA we obtained twenty bands on agarosegel with the same molecular weight. Sequencing of all theamplicons were high similar. Compared with those available inthe database, they also revealed best match BLAST with the same

Fig. 4. Cleistothecia observed in DG18 agar plate (bar ¼ 200 mm).

E. halophilicum (An. A. halophilicus) (GenBank ID: [EF652088]), at97% of similarity. Ten sequences were deposited at the NCBIdatabase (Table 1).

4. Discussion

Fungi damaging materials in indoor environments are chieflyprimary colonizers capable of rapid growth even when wateractivity is low (i.e. Aw <0.8). When a substrate is attacked bya fungus, its water activity changes sufficiently to support thegrowth of other species (fungi and bacteria), such as in naturalsuccessions (Samson et al., 1994). Secondary colonisers are speciesthat have a higher resistance to water stress. These species developthanks to unstable microenvironments characterised by smallchanges in air temperature or humidity due to night/day alterna-tion. Poor ventilation and surface temperature dynamics canproduce foci of water condensation and local micro-climates withlocalized peaks of Aw greater than the surrounding indoor

Fig. 6. Optical micrograph on adhesive tape: active conidiophore and conidia stainedwith FDA (bar ¼ 20 mm).

Fig. 7. SEM micrograph of fungal elements recovered on adhesive tape: conidia and hyphae (bar ¼ 2 mm ); B: conidial head, conidia and hyphae (bar ¼ 10 mm).

Fig. 8. SEM micrograph of mycelium growing in pure culture on agar medium,showing a portion of an hypertrophic hypha with bare and short hairs (bar ¼ 1 mm).

M. Montanari et al. / International Biodeterioration & Biodegradation 75 (2012) 83e88 87

environment. These circumstances are favourable to some fungalspecies that are able to proliferate in places where the overallenvironmental conditions would otherwise appear to be hostile(Pinzari, 2011). Findings reported here, are consistent with singlespecies E. halophilicum contamination of materials stored in Com-pactus shelves. Identification of this fungus was achieved bya combination of conventional, molecular methods and SEMobservations. Isolation of this fungus was achieved only by directplating on DG18 agar of fungal elements collected directly from

Fig. 9. SEM micrographs fungal growth from pure culture on agar me

volume surfaces. The sterile nitrocellulose membrane techniquehas shown to be very effective for collection of fungal elements fordirect plating. All sequences obtained from pure cultures werehighly similar to sequences obtained from total DNA extracteddirectly from the adhesive tape samples (environmental samples),and all these sequences had the best BLAST matches (97e100%similarity) with the unique rDNA sequence attributed toE. halophilicum as published on GenBank Data Base (Peterson,2008).

It is important to emphasize that, in the present study, thefungus showed a different life cycle and morphological aspectin vivo and in vitro. On volume bindings the fungus was observedonly its anamorphic stage as conidiophores and conidia. Whenobserved at SEM, conidiophore stipes and vesicles were denselycoated by curly micro-filaments similar to hairs all over theirsurface. On agar media, the fungus presented in its teleomorphproducing many ascomata and ascospores, SEM observationsshowed the presence of bare and short hairs on hypertrophichyphae. These features of anamorphic and teleomorphic structuresare consistent with those described by Christensen et al. in theoriginal description of E. halophilicum (1959) and by Samson andLustgraaf (1978). However, the dense layer of hairy and curlystructures observed on the wall of conidiophores and hyphae werenever reported before. Hairs might be an adaptation of the fungusto increase its ability to capture water from the surrounding air ina dry environment. E. halophilicum is a xerophilic fungus witha high tolerance to water stress. The minimum observed wateractivity (Aw) for germination and growth is 0.675, one of the lowestfor Eurotium species (Christensen et al., 1959). The occurrence ofthis fungus is associated with air-dust (Abdel-Hafez et al., 1990) orhouse-dust in association with mites and Aspergillus penicillioides

dium. A: ascomata (bar ¼ 100 mm); B: ascospores (bar ¼ 10 mm).

Table 1Nucleotide sequence accession numbers at NCBI database.

Code Accession number

Isolate MM373 [JN839940]Clone 1m1 [JN839949]Clone 2m1 [JN839950]Clone 7m1 [JN839951]Clone 8m1 [JN839952]Clone 9m1 [JN839953]Clone 12m1 [JN839954]Clone 13m1 [JN839955]Clone 14m1 [JN839956]Clone 17m1 [JN839957]Clone 22m1 [JN839958]

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and storage of dry food (Hocking and Pitt, 1988). Sequences of E.halophilicum have been associated with library material only byMichaelsen et al. (2010) by DGGE-fingerprinting, without anyinformation about its viability and role on the substrate deterio-ration. Therefore this is, in our knowledge, the first report whereE. halophilicum is isolated and cultivated from book bindings andwhere its role in the contamination of library materials is demon-strated by a combination of visual, culture and culture-independentmethods. Similar contamination patterns and microscopic featuresobserved in several samples collected with the adhesive tapetechnique during previous surveys performed in other archives andlibraries where Compactus type shelving is used (Montanari et al.,2007; Pinzari and Montanari, 2011) suggest that E. halophilicummight have a large distribution in this particular environment, atleast in Italy. Detection failure in previous surveys may have beendue to inadequate sampling procedure and unsuitable agar media.As suggested by Christensen et al. (1959) and Samson and Lustgraaf(1978), the occurrence of this fungus in indoor environments maybe underestimated due to inadequacy of classical methods and itsvery slow growth on typical media.

In conclusion the high tolerance to water stress combined withthe high affinity to library materials showed by this fungus repre-sent a serious threat for librarians and conservators. Conventionalcontrol of temperature and relative humidity in conformity withrecommended standards may be insufficient to prevent materialcolonization, especially if enclosed systems with low ventilationrate, such as Compactus type shelves, are adopted.

The operations suggested to conservators and librarians to avoidthe diffusion of this fungus are: i) the HVAC (heating, ventilation,and air conditioning), if available in the library, must be checked forefficacy, and ventilation grilles cleaned and checked to verify theircorrect function, ii) HVAC filters should be changed frequently; iii)to facilitate ventilation, side panels of the Compactus units shouldbe removed or a program of daily stack moving should beestablished.

Frequent visual inspection of materials stored within Compac-tus shelving units is highly recommended, notwithstanding thepresence of a well-functioning HVAC and maintenance of optimalstandard climatic values. Micro-environmental anomalies andlocalised deterioration phenomena, if promptly detected can beeasily contained and resolved, while invasive reclamation/rehabil-itation of materials should, as far as possible, be avoided andimplemented only as a last resort.

Further studies have now been established in Italian librariesand in our laboratories for E. halophilicum ecology, metabolicrequirements, and damage actually provoked on books.

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