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International Biodeterioration & Biodegradation 98 (2015) 121e130

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International Biodeterioration & Biodegradation

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The assessment of fungal bioaerosols in the crypt of St. Peter inPerugia (Italy)

L. Ruga, F. Orlandi*, B. Romano, M. FornaciariDepartment of Civil and Environmental Engineering, University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy

a r t i c l e i n f o

Article history:Received 22 September 2014Received in revised form18 December 2014Accepted 23 December 2014Available online

Keywords:Aerobiological monitoringCultural heritageIndoor environmentCryptFungal sporeBiodeterioration

* Corresponding author. Tel.: þ39 075 5856067; faE-mail address: fabio.orlandi@unipg.it (F. Orlandi)

http://dx.doi.org/10.1016/j.ibiod.2014.12.0100964-8305/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The inspection of the quality of the indoor air in which a work of art or a historical artefact is keptbecomes essential for its conservation. The determination of organic pollutants represents an importanttool in pre-emptive conservation. The study investigated the quality of the air in the crypt of the Basilicaof St. Peter in Perugia (Italy) through different methodologies. The objectives included the analysis of thelevels of biological particulates of fungal origin, and the determination of the degree of variability of theairborne spore concentrations, as indicative of the level of contamination of the environment. Thequantitative analysis of the airborne fungal component demonstrates that across the whole periodconsidered there were wide variations in the bioaerosols, heterogeneous spore distributions anddifferent peak concentrations in the areas studied. The qualitative analysis of the airborne fungalcomponent allowed the determination of the different fungal genera present, both in the interior of thecrypt and in the outside environment. The analysis of the data shows an increasing trend over the periodconsidered, with the highest values during the months of June and July.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

The conservation of our cultural heritage has for many yearsfavoured protection against the risk of degradation, to limit anydamage before it gets worse (‘pre-emptive conservation’). In thisway, the focus of the conservation moves from the work of art itselfto the physical environment in which it is kept (Getty ConservationInstitute, 1994). The regulation of the quality of the indoor air inwhich awork of art or a historical artefact is kept becomes essentialfor its conservation. In particular, determination of organic pollut-ants is useful, so that the procedure to optimise the environmentalprotection represents an important tool in pre-emptive conserva-tion, and above all allows the definition of the levels of biodeteri-oration risk (which arises from the life processes of livingorganisms that produce changes in the materials of their sur-rounding environment) and of sanitary hygiene for the associatedoperators and visitors (Sorlini, 1993; Mandrioli and Caneva, 1998;Urzì et al., 2001; Pinna, 2003; Aira et al., 2007; Angelosante et al.,2007; Ruga et al., 2007, 2008; Chen et al., 2010). The methodolo-gies and the operational techniques for the prevention and recov-ery of damages caused by the biodeterioration improve the

x: þ39 075 5856598..

information about the biological risk factors of the workers. Thesediseases may be particularly dangerous and related to the conser-vation and restoration of ancient artefacts often degraded by theaction of living organisms. The numerous examples of diseases thataffect the restorers may be caused by the manipulation of artefactsfrom infected areas or environments, or by artefacts that act asvectors of pathogenic agents not particularly dangerous in thebroad sense, but which have been isolated for a long period, andagainst which the human body struggles to develop specific im-munity. In the confined environments where particularly valuablehistorical, artistic or cultural objects are kept, such as museums,libraries, archives, churches and hypogea, there are a wide range ofmicroorganisms andmacroorganisms that represent potential risksfor the degradation of these objects. These can also act in synergywith other factors, such as the atmospheric conditions, the natureof the artefact, its location within the environment, the materialsthat can be subject to attack, and any physicochemical degradationprocesses that are already present (Ruga et al., 2008).

Moreover, an artefact that has been previously restored, it maysometimes be subject to an increased susceptibility to biologicaldegradation due to the type of substances used in recovery oper-ations (Gu, 2003; Cappitelli et al., 2004).

In confined environments, there is a lack of the natural circu-latory mechanisms that are normally part of the external envi-ronment, which results in greater amounts and settling of dust and

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130122

potentially contaminating material. These can arise from faulty airconditioning systems, from the presence of people, who representone of the major carriers of microorganisms, and from sources thatare in direct contact with the external environment, such as en-trances and exits, or windows or doors (Orlandi et al., 2011; Quianet al., 2012; Gauzere et al., 2014). These airborne particles can bedeposited on the works of art, and under favourable conditions oftemperature, humidity, light and substrate, they can use thedifferent surfaces not just as a physical support for their growth, butalso as a nutrient source. This can cause irreversible structuralchanges or damage to such protected works of art, with a loss ofstrength and structure, and also changes from an aesthetic point ofview (Lopez-Miras et al., 2013). To avoid irreversible deteriorationof such artefacts, and to evaluate the indoor biological risk, it isimportant to periodically carry out aerobiological monitoring of thebiological components of the air of their environment, which pro-vides the characterisation of the air quality from a biological pointof view, both quantitatively (e.g., degree of contamination) andqualitatively (e.g., presence of potentially biodeteriogens species orwith potential allergenicity) (Gravesen, 1979; Kurup et al., 2000).This allows the identification of any contaminant sources and thecarrying out of suitable interventions (Nugari and Roccardi, 2001;Ruga et al., 2008). The Aerobiology represents an important toolfor the preventive conservation and the public health. Moreover, itallows to define important knowledge for operators involved in theartefacts conservation and restoration, providing also importantinformation in terms of hygienic, safety and health protection. Inenvironments such as crypts, with typical characteristics of theunderground environment, alterations to the surfaces can arisefrom the development of either photoautotrophic microorganismsin the presence of natural or artificial light, or of heterotrophicmicroorganisms when there are organic substances, even in smallquantities. In particular, wall paintings, such as murals or frescos,are usually poor in organic substances, and the development ofheterotrophic microorganisms is conditioned not only by micro-climatic factors, but also by the biological pollutants present in theair that arise from the propagation of biodeteriogens or organicsubstances that represent sources of food for manymicroorganisms(Nugari et al., 2007). In this way, such artefacts can suffer fromdegradation, and the problem of biodeterioration arises when livecells of biodeteriogens microorganisms are deposited on the sur-faces. Under favourable conditions of temperature and humidity,heterotrophic microorganisms can be sufficiently able to developand multiply, such as the bacteria and fungi that can be found inminimal dust deposits or organic residues that are generated by theprimary and secondary autotrophic colonisers (Nugari et al., 1993;Saarela et al., 2004). Microscopic fungi, in particular, are the mostimportant factors of all of the biological changes that frescos canundergo, because with their nutritional needs, their reactions to-ward external stimuli, and their ability to adapt to often adverseenvironmental conditions, microscopic fungi can show great di-versity and great metabolic activity (Ciferri, 1999; Gorbushina et al.,2004; Zammit et al., 2009; Pangallo et al., 2012; Sterflinger andPi~nar, 2013; De Leo and Urzì, 2014). Their metabolic productscause further chemical damage (Beech, 2004). The capacity of fungito dissolve carbonates depends on available carbon sources, such asoxalic and citric acids which may mobilize cations with chelatingactivity (Hirsch et al., 1995; Wollenzien et al., 1995; Milanesi et al.,2006). Fungal organisms, for the large capacity of metabolicadaptation, can colonize and use as a nutritional substrate alsosynthetic polymers (Lugauskas et al., 2003). In presence of somepolymers in which the organic fraction is absent the fungi devel-opment is linked to the organic additives present in the siliconeresin used to protect and consolidate stone materials (Cappitelliet al., 2007).

Therefore, the aim of this study was to investigate, throughdifferent methodologies, the presence of airborne microrganisms,potentially biodeteriogens for the stone artefacts in a semi-underground environment.

In particular, the study objectives included: characterisation ofthe quality of the air in the crypt of the Basilica of St. Peter inPerugia (Italy) through analysis of the levels of biological particu-lates of fungal origin, and determination of the degree of variabilityover time of the airborne spore concentrations, as indicative of thelevel of contamination of the environment; analysis of the qualityof the sampled particulates, and determination of airborne fungalgenera; identification of possible relationships between the insideand outside concentrations of the particulates, and the microcli-matic conditions, particularly for the temperature and relativehumidity of the air.

Materials and methods

Studied area

This study was carried out in the conservation and exhibitionareas of the crypt of the Basilica of St. Peter in Perugia (property ofFoundation for Agricultural Education in Perugia, Italy), situated ata depth of 3 m below the floor of the church of St. Peter. In 2000some measures were taken to consolidate the plasters and thefresco painting with acrylic resins. A special care was required bythe disinfection of painted surfaces, affected with deteriogens or-ganisms (algae), realized through biocide applications. The crypt isoriented northeast to southwest, following the same orientation ofthe church above (Fig. 1a), and it can be accessed down a ladderplaced at the bottom of the left aisle, which ends in the first section.From here, there is a corridor, and running along its south side thereare five brick niches, forming apses of equal size (Fig. 1b). On thenorth side, there is access to an exedra (Fig. 1c). The corridor con-tinues along to the final part of the crypt, where there is a final areaof equal size to that at the entrance to the crypt.

Aerobiological sampling

The objectives of this study were achieved mainly through theairborne fungal particles monitoring. The monitoring has beendone with three different methods. The sampling was defined inrelation to the environments that would be more critical and theareas in which there might be higher risk of biodeterioration (e.g.,the parts located near to the entrances and exits, the areas char-acterised by the wall paintings). In particular, the sampling forairborne spores was carried out: in the first area (referred to as the‘entrance’), as this was closer to the entry and exit from the crypt; inthe ‘exedra’, as this was the area closest to the frescos and con-nected with the church above through three little holes(15 � 10 cm) in the ceiling; and in the final area (referred to as the‘internal area’), as this was considered to be the innermost part ofthe crypt. Fig. 1a shows the plan of the crypt where the monitoredareas are indicated. The sampling was conducted on a weekly basisfrom March to July, 2011.

The monitoring was realized during the same morning periodutilizing contemporary the three sampling methods.

Fungal particles countingThe simple counting of particles gives a first “quick and dirty”

approximation of a microbial contamination of the air. For thesampling of the airborne fungal particles, a Personal Volumetric AirSampler spore trap for glass slides was used (Burkard CompanyLtd.). This was based on the original Hirst model (1952), and itoperates on the principle of impact for depression, through the

Fig. 1. The crypt (picture A. Franken, from Palombaro, 2007). (a) Plan of the crypt,showing the sampling areas: entrance, exedra, internal area. (b, c) Photographs takenfrom the corridor within the crypt, looking along the south side at two of the five brickniches that form apses of equal sizes (b), and into the central apse of the exedra (c),showing three of the seven arches decorated with a wolf-teeth design, and three of thecolumns with alternating red and yellow bands. The altar positioned on two half-circlesteps by the central wall can also be seen (c).

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130 123

aspiration of nominal air volumes, and provides data on the fungalspore concentration (number of spores/m3). The spores identifiedwith optical microscopy were converted into the number of sporesper cubic meter of air sampled through a conversion factor basedfirstly on the ratio between the observed and the total areas of thesampling slide (considering themicroscopy optical field on the baseof the amplification utilized) and secondly on the air volume pertime unit sucked by the instrument (Ogden et al., 1974).

The captured atmospheric air (10 L/min) was conveyed to anadhesive slide with silicone oil. The sampler was positioned, one

time in a week for 10 min (for a total of 0.1 m3 of sampled air), bothin the interior of the crypt and in the external environment of theBasilica, far from vegetation.

This study also examined the indoor environment of the crypt,as a comparison with the outdoor environment of the Basilica(referred to as ‘outside’). As some of any biological polluting agentspresent might come from the outside environment, the outdoorpollution also needs to be taken into consideration during theevaluation of the risk factors (Mandrioli et al., 2007).

Cultural analysesFor the analysis of the airborne viable fungal particles, two

sampling systems were used: a passive and an active method. Thepassive sampling of the biological particles used the impact-sedimentation technique on Petri dishes (Ruga et al., 2008). Thissimple method was used to contemporaneously monitor thedifferent places. Thiswas carried out in the indoor spaces chosen formonitoring, with the exposure to the air one time in aweek, for 1 h,somePetri dishes (90mmdiameter) containing SabouraudDextroseAgarwith chloramphenicol, in order to limit bacterial growthwhichmay hinder the growth of fungal colonies (Booth, 1971). The activesamplingusedone time in aweek a three-stageAndersen (first stage>7.0 mm; second stage between 3.3 and 4.7 mm; third stage between1.1 and 2.1 mm) type impact sampler (Sorlini, 1993; Nugari, 2003).During the sampling phase, the samplerwas placed in the individualareas selected for themonitoring. The time for the air aspirationwasagain set to 10min (at 28.3 L/min) determining a total of 0.283m3 ofsampling air perweek. The analysis of the airborne viable culturablefungal component has been carried out only in the indoor spaces.The comparison of indoor and outdoor fungal component vitalitywas not realized because the real objective of work was to evaluatethe vitality of spores in the crypt, considering that the scientificliterature (Mandrioli and Caneva, 1998) suggests as in outdoorspaces the atmospheric pollutants, depending by their nature andconcentration, should stimulate or inhibit the fungi metabolisminfluencing the spore vitality.

Identification of fungal groupsThe Fungi identification was realized on the base of colonies

macro-morphological and spore micro-morphological description.Spore reference images, Atlas and spore collection were used forthe spore identification (S�aenz Laín and Monserrat Guti�errezBustillo, 2006). In all the considered methods the “fungal strains”were classified to genus level. The material sampled directly on theslide with the Personal Volumetric Air Sampler spore trap wasstained with lactophenol blue solution and observed under opticalmicroscope (magnification 400�).

Thematerial sampled on Petri dishes was incubated at 28 ± 2 �C.After 3 days and 6 days of incubation, the microbial colonies thatdevelopedwere counted (Ruga et al., 2008). For the passivemethodthe data obtained are expressed as colony forming units per unitarea (CFU 100 cm�2), for the active method as CFU per unit volume(CFU m�3 of the air analysed). In both airborne viable fungalcomponent sampling methods, the identification was preceded bystereo-microscope observation of colonies; successively the slideswere prepared with lactophenol blue solution and observed underoptical microscope (magnification 400�).

Measurement of temperature and relative humidity

The study of the microclimatic conditions in underground en-vironments is a prerequisite for an understanding of the causes ofany degradation, and to define the microbial diffusion processes toidentify the most appropriate conservative mode (Monte andFerrari, 1993; Nugari et al., 1998, 2007; Pietrini et al., 2005).

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130124

To obtain a more complete picture of the environmental con-ditions some variables (temperature and relative humidity) wereconsidered inside the crypt contemporary to the aerobiologicalsamples and in outdoors areas.

The considered environmental variables were measured usingthe digital thermo-hygrometer (RTGR328N Thermo/Hygro Sensor,Oregon Scientific), cable free, RF frequency 433 MHz. The range oftemperature measurement is from �20 �C to þ60 �C, the range ofmeasurement of relative humidity is between 20% and 99%.

Statistical analysis

The curves for the relative concentrations in the individual areasmonitored and for the external environment were constructed onthe basis of the data for the monthly averages. To identify possiblerelationships between the concentrations of the indoor and out-door airborne total fungal particles with the temperature and therelative humidity, the monthly mean values were analysed statis-tically, using correlation tests (Pearson linear coefficients).

Results

Quantitative and qualitative analyses of the total fungal particlescounting

The quantitative analysis of the airborne fungal particles dem-onstrates that during the whole period considered there were widevariations in the bioaerosols, spores heterogeneous distributions,and different peak concentrations in the areas studied.

The overall mean fungal concentrations were higher in the vi-cinity of the area of the entrance to the crypt, with 1862 sporesm�3,than in the area of the exedra, with 973 spores m�3. These levelsincreased again towards the internal area, with a mean concen-tration detected in the air of 1274 spores m�3. In Fig. 2, these dataare shown as the general trends of the monthly mean concentra-tions of the total fungal spores that were recorded within each ofthese different areas of the crypt, and for the outside monitoring,over this period from March to July, 2011. For better interpretationof the data, these values are illustrated on a logarithmic scale charton which also the real mean concentration values are reported. Asmentioned above, the entrance area is the first room of the sam-pling series. In this area, the monitoring data show some variabilityover the period of investigation. The highest concentration of theairborne spores was in June, with 7223 spores m�3, which thus

1

10

100

1000

10000

Mar Apr

Log [c

once

ntra

tion]

(spo

res m

-3)

Entrance Exedra

298

280

323

608

183

247

647

177

175

Fig. 2. Comparison of the monthly mean total fungal spore concentrations for the variouconcentrations are reported as spore m�3).

represents the highest mean level among the indoor samplingareas over the entire period of investigation (Fig. 2). Themonitoringof the exedra, which is the area with the paintings, and thereforethe closest part to the frescos, also showed wide variability ofspores over these several months of sampling. Here, the highestmonthly mean concentration was in July, with 2215 spores m�3

(Fig. 2). For the analysis of the final part of the sampling series, asthe internal area, there was again variability in the airborne sporeconcentrations through the analysis period, with the highestmonthly mean in June, of 3908 spores m�3, parallelling that of theentrance area peak (Fig. 2). As for these indoor areas, the aero-biological monitoring for the outdoor area showed large variabilityfor these bioaerosols over the study period. Here, for the months ofMarch, May and July, the recorded mean values were higher thanthose found indoors, with the peak levels reached in July, with1870 spores m�3 (Fig. 2). The qualitative analysis of the airbornefungal component allowed the determination of the differentfungal genera present, both in the interior of the crypt and in theoutside environment. Table 1 lists the fungal genera that wereidentified in the different areas, along with their absence or pres-ence for each of the analysis areas. Cladosporium and Penicilliumand/or Aspergillus (these genera were generally combined, aswithout fruiting bodies it was difficult to define to which group thespores belonged, as they are very similar to each other) were themost represented genera that were more frequently recorded inthese specific areas, and they were present in all of the measure-ments, regardless of the seasonal trend. Within the crypt, the in-ternal area showed the widest variety of fungal spores, in terms ofthe different genera. Moreover, some of the fungal genera that werenot detected in the outside environment were recorded inside thecrypt, including in particular: Arthrinium, Gymnopilus, Phaeos-phaeria and Polythrincium. Some of the genera in the outsideenvironment were confirmed within some of the areas of the crypt(e.g., Amphisphaeria and Laccaria), while others had not spread intothe crypt (e.g., Caloplaca, Claviceps, Exosporium, and Sporidesmium).Conversely, Cladosporium spp. were present in all of the measure-ments, both within and outside the crypt.

Quantitative and qualitative analyses of the viable airborne spore

The data obtained with the impact-sedimentation method onthe Petri dishes also showed that the total fungal quantities werevery variable in each of the monitored areas and across the indi-vidual months considered (Table 2A). In this sampling series in the

May Jun Jul

Internal area Outside

175

360

860

7223

1948

3908

1003

1430

2215

1135

1870

s areas within and outside the crypt (histograms with log value, at the base the real

Table 3Fungal genera identified in the air samples using the sedimentation (A) and theAndersen (B) sampling methods in the various areas within the crypt, for the periodof March to July 2011.

Genus Entrance Exedra Internal area

A)Acremonium þ þ �Alternaria þ þ þAspergillus þ þ þBotrytis � þ þChaetomium � � þCladosporium þ þ þGeotrichum þ þ �Humicola þ þ þ

Table 1Fungi genera identified in the air samples collected with the Personal Volumetric AirSampler spore trap in the various areas within and outside the crypt, collectively forthe period of March to July, 2011.

Genus Crypt sampling area Outside

Entrance Exedra Internal area

Agaricus þ � þ þAgrocybe � � þ þAlternaria þ þ þ þAmphisphaeria � þ � þArthrinium þ þ þ �Bipolaris þ � þ þBotrytis þ þ þ þCaloplaca � � � þCerebella � � þ þChaetomium þ þ þ þCladosporium þ þ þ þClaviceps � � � þCoprinus þ þ þ þCurvularia � þ þ �Epicoccum � þ þ þExosporium � � � þFusarium � þ � þGymnopilus þ þ � �Laccaria þ � � þLepiota þ � þ þLeptosphaeria þ þ þ þMassaria � � þ �Myxomycete � � þ �Oidium � þ � �Penicillium/Aspergillus þ þ þ þPericonia þ þ þ þPeronospora þ þ þ þPhaeosphaeria þ � � �Pithomyces þ þ þ þPleospora þ þ þ þPolythrincium þ � þ �Pseudocercospora � þ � �Puccinia � � þ �Scopulariopsis þ þ þ þSporidesmium � � � þStemphylium þ � þ þTorula þ � þ þXylaria þ � þ þ

The Penicillium/Aspergillus group includes both of these genera, as without fruitingbodies it was difficult to define to which group the spores belong, as they are verysimilar to each other.

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130 125

crypt, the greatest mean fungal burden was in June (117 CFU100 cm�2). As indicated above, this confirmed again the high levelsfor June, as for these samples, June was also the period with thegreatest presence of airborne particulates. Over the whole of the

Table 2Total fungal load for each sampling area, calculated using the sedimentation (A) andthe Andersen (B) sampling methods.

Sampling area Monthly mean fungal load (CFU cm�2)

March April May June July Mean value

A)Entrance 13 13 12 110 95 49Exedra 27 11 11 94 66 42Internal area 42 36 35 146 115 75

Mean 27 20 19 117 92

Sampling area Monthly mean fungal load (CFU m�3)

March April May June July Mean value

B)Entrance 214 105 138 648 529 327Exedra 186 124 90 757 550 341Internal area 336 365 325 650 729 481

Mean 245 198 185 685 602

sampling period within the crypt, the greatest number of fungalcolonies was in the internal area (Table 2A).

The qualitative analysis of air samples obtained by the impact-sedimentation method also allowed the determination of thepresence and viability of spores from the different fungal genera inthe individual areas of the crypt (Table 3A). A consideration of thesedata confirms the presence of some genera already detected withthe Personal Volumetric Air Sampler spore trap, such as Alternaria,Botrytis, Cladosporium, Chaetomium and Scopulariopsis. This methodalso allowed the detection of the spread of the fungal genera forthose that were not found in the total fungal spores monitoredwiththe previous method, such as the genera Acremonium, Geotrichum,Humicola, Laccaria and Paecilomyces. The genera represented andmonitored more frequently were Aspergillus, Penicillium and Sco-pulariopsis. For the distribution of the fungal genera within thestudy areas, this was predominantly uniform. Table 3A shows thedistributions by area of the fungal genera recognized through thisperiod of investigation. The Andersen sampling allowed furtherinvestigations here, with the detection of larger numbers ofairborne spores (Table 2B). These data show that also in this case,the total fungal quantities were very variable in each of the moni-tored areas and across the individual months considered. Overall,the highest mean concentration of viable airborne spores was inJune (685 CFU m�3), confirming the monitoring data indicatedabove. Here again, the combined data across the sampling periodshowed the greatest number of viable fungal spores (as CFU) in theinternal area (Table 2B), further confirming the findings with themonitoring method indicated above. Also in this case, the qualita-tive analysis of the air samples obtained with the Andersen sam-pling method allowed the detection and identification in the

Laccaria � þ þLepiota � � þPaecilomyces � � þPenicillium þ þ þPenicillium/Aspergillus þ þ þScopulariopsis þ þ þB)Acremonium þ � �Alternaria þ þ þAspergillus þ þ þBipolaris þ � �Botrytis � þ �Cladosporium þ þ þGeotrichum þ þ þLaccarla þ � þLepiota � � þPaecilomyces þ � þPenicillium þ þ þPenicillium/Aspergillus þ þ þScopulariopsis þ þ þTrichoderma þ � þ

The Penicillium/Aspergillus group includes both of these genera, as without fruitingbodies it was difficult to define to which group the spores belong, as they are verysimilar to each other.

Table 4Monthly mean temperature and relative humidity in the various areas within andoutside the crypt, for the period of March to July 2011.

Sampling area Monthly mean Overallmean

March April May June July

Temperature (�C)Entrance 14.9 16.7 18.2 21.0 22.2 18.6Exedra 14.8 16.1 17.2 19.8 21.5 17.9Internal area 14.8 16.0 17.1 19.6 21.3 17.7Outside 17.3 16.8 19.2 22.9 24.6 20.1

Relative humidity (%)Entrance 52 54 61 70 71 62Exedra 58 64 69 76 77 69Internal area 59 65 71 80 80 71Outside 47 42 50 50 63 50

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130126

individual areas of the crypt of the viable spores belonging to thedifferent fungal genera, as potential biodeteriogens (Table 3B). Inparticular, these data confirmed the presence and vitality of some ofthe genera already detected with the Personal Volumetric AirSampler spore trap and the passive sampling method, includingAlternaria, Cladosporium and Scopulariopsis. This method alsoallowed the detection of the spread of Trichoderma spp., a generathat was not found in the fractions monitored by the abovemethods. This qualitative analysis of air samples taken using theAndersen instrument showed that the fungal spores that weremostfrequently sampled were those of the genera Aspergillus, Penicilliumand Scopulariopsis. The distribution of the fungal genera within thecrypt study areas did not show any significant differences, with amainly uniform distribution seen. Table 3B gives the distribution byarea of the fungal genera that potentially represent biodeteriogensthat were recognized through the period of investigation.

Micro-climatic conditions

Table 4 gives the data related to the temperature and relativehumidity that were recorded and then reported on ameanmonthlybasis, both within the crypt and in the outside environment, andalso as the means for the entire study period considered (March toJuly, 2011). These temperatures and relative humidities showedoverall increasing trends during this period, which was probablydue to the seasonal trends. The internal temperatures recorded inthe three areas of the crypt across the fivemonths of the studywerevery similar. There was, however, wider variability seen for therelative humidity across the monitored areas of the crypt for thestudy months (Table 4).

Relationships between total airborne spore concentrations and thetemperature and relative humidity variables

Pearson correlation analysis presented in Table 5 (p < 0.05 sig-nificant values as undersigned) allowed evaluation of the possible

Table 5Pearson correlation values. Undersigned values are significant at p < 0.05.

Entrance Exedra Internal area Outside Crypt

Entrance 1.00 0.67 0.99 0.25 0.99Exedra 0.67 1.00 0.69 0.81 0.78Internal area 0.99 0.69 1.00 0.24 0.99Outside 0.25 0.81 0.24 1.00 0.37Crypt mean 0.99 0.78 0.99 0.37 1.00Temperature 0.54 0.92 0.57 0.85 0.65Humidity 0.65 0.89 0.67 0.78 0.74T�C outs. 0.58 0.95 0.59 0.92 0.69R.H. (%) outs. 0.15 0.77 0.15 0.99 0.28

relationships between the concentrations of the indoor and out-door airborne spore particles and the microclimatic conditions, interms of the temperature and relative humidity of the indoor andoutdoor air. The analysis of these data revealed a high correlationbetween the concentrations recorded in the outside monitoringand only the intermediate area in the sampling series in the crypt.In particular, the exedra showed a highly significant correlationwith the outside, with r ¼ 0.81. This high correlation value almostcertainly can be related with the presence of four apertures(15 cm � 20 cm) which connect the underground space (exedra)with the Basilica constructed above determining therefore air flowsbetween underground and overground sites. From the point ofview of the relationships between the two indoor monitoredenvironmental variables (temperature and relative humidity) andthe concentrations of spores detected, it is important to note thatthe highest and significant correlations were carried out onlyconsidering exedra area (Table 5). Here, it was possible to note adirect relationship between an improvement in the favourableconditions for the proliferation of fungal spores (increased tem-perature and relative humidity) and the average concentrations ofthe spores themselves respectively r ¼ 0.92 and r ¼ 0.89.

Finally, the correlation analysis showed a significant relation-ship between the temperature and relative humidity within thecrypt. Considering the temperatures recorded inside and corre-lating these with the relative humidity, again inside the crypt, therewas a statistically significant relationship seen, as expressed by apositive correlation coefficient of r ¼ 0.98.

Discussion

The qualitative and quantitative monthly analyses of theairborne fungal component allowed the identification of the pres-ence of different fungal species, and different concentrations oftheir spores in the environments within, and outside, the crypt. Forall the airborne fungal particles, the analysis of the data shows anincreasing trend of their presence over the period considered, withthe highest values during the months of June and July. This increaseis probably related to the seasonal trends. In geographical areassuch as Perugia, which has a transitional character from aTemperate to a Mediterranean climate (Rivas-Martinez, 1996), thepeaks for the maximum concentrations of fungal spores normallyoccur in summer and autumn, when the relative humidity is at itshighest (Montacutelli et al., 2000). The occurrence here of themaximum concentrations in June and July corresponds to databroadly demonstrated in other similar studies: the growth of fungalspecies is favoured by an air temperature range from 18 �C to 32 �Cand high relative humidity, parameters that are often observedduring the summer season in the region of the crypt of the Basilicaof St. Peter in Perugia, Italy (Pelizzari, 1996; Pyrri and Kapsanaki-Gotsi, 2007). The analysis of the total fungal particles allowed themeasurement of the trend of the airborne concentrations of spores,as an indication of the degree of contamination of the

mean Temperature Humidity T�C outside R.H. (%) outside

0.54 0.65 0.58 0.150.92 0.89 0.95 0.770.57 0.67 0.59 0.150.85 0.78 0.92 0.990.65 0.74 0.69 0.281.00 0.98 0.97 0.830.98 1.00 0.95 0.740.97 0.95 1.00 0.880.83 0.74 0.88 1.00

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130 127

environments in the crypt. The analysis of the airborne viablefungal spores allowed the determination of the specific presence ofdifferent fungal genera that represent potential biodeteriogens. Thepassive method, applied through the technique of impact-sedimentation on Petri dishes, allowed the achievement of infor-mation on the part of bioaerosols that can be deposited undergravity onto a surface. This provides indications of the levels ofcontamination in the environment and the depositing risk to ar-tefacts maintained in the crypt (Pasquarella et al., 2011). Themethod of active sampling, which was carried out through the useof the Andersen instrument, allowed the measurement of theconcentrations of particles in the air that can be deposited bysedimentation or other mechanisms, providing, in particular,quantitative data on the viable suspended microflora. The use ofonly one method may underestimate the diversity and the con-centration of certain fungal propagules for several reasons (Pyrriand Kapsanaki-Gotsi, 2007). The three methods used in this studyallow to have more information about indoor airborne spore. In thestudy of biodeterioration of mural paintings, the impact-sedimentation method with Petri dishes may be useful in indoorspaces. Although this method does not provide quantitative data(the sampled air volumes are not known) is useful in investigationrealized in stable environmental conditions, such as in hypogea(Nugari and Roccardi, 2001). Moreover, the fungi that cause dete-rioration of mural paintings principally belong to the Deuter-omycetes generally with spores greater than 2 mm (Nugari et al.,1993). Particles of these dimensions undergo gravitational sedi-mentation quite efficaciously in calm air conditions (Camuffo andBernardi, 1985). The Andersen method has been found more suit-able for cultural heritage being suggested as international standardfor this type of analyses (Nugari, 2003). Moreover, the analysesrealized with the volumetric methods (Andersen's sampler andBurkard air sampler for glass slides) allow to complete the quan-titative information and to have comparable results (Nugari andRoccardi, 2001). Environments such as crypts, which are generallynot connected directly with the outside at the time of their dis-covery, are characterised by structures andmicroclimates that havenot been significantly modified or altered. This process of modifi-cation then occurs when an environment thatmight have remainedisolated for many years is once again in contact with the externalatmospheric conditions. These conditions also depend on the areaof discovery; indeed, in several cases, such underground chambershave been characterised by a slight trend in the daily and seasonalclimate (Monte and Ferrari, 1993). The level of risk of the phe-nomena of biodeterioration is indeed related to the microclimatepresent in such crypt-like environments, and it varies mainly inrelation to temperature, humidity, and light. Therefore, it can beseen that the relationship between these values and the conditionsin both the internal and outside environments can inducemicrobialgrowth that can be very dangerous for conservation activities.Environmental conditions that provide a relative humidity over60%, a temperature between 10 �C and 30 �C and poor ventilation,which are typical of environments such as crypts, are all featuresthat are particularly favourable for fungal growth (Nugari et al.,1993; Cataldo et al., 2005).

In addition, the chemosynthetic microbial (e.g., bacteria) andheterotrophic (e.g., fungi) colonisers might not reveal their pres-ence under normal visual examination due to their small size,although can still be active on the artefacts, through the formationof crusts, dust and layers (Giacobini et al., 1987; Ciferri, 1999;Sterflinger and Pi~nar, 2013; Onofri et al., 2014).

The fungi are able to penetrate into the rock material by hyphalgrowth and by biocorrosive activity, due to the excretion of organicacids or by oxidation of mineral-forming cations, preferably ironand manganese. Predominately strains of Exophiala, Penicillium,

Aspergillus, Cladosporium, Alternaria, Aureobasidium, Ulocladiumand Phoma have been isolated. Their deteriorating activity includesdiscolouration of stone surfaces, due to the excretion of melaninsby dematiaceous fungi, and mechanical stress to stone structures(Warscheid and Braams, 2000). Penetration of hyphae from fungiand lichens also induce stresses in calcareous rocks (Caneva et al.,2002).

A first observation of considerable interest in terms of thegrowth of fungi on the frescos is that it evolves in stages: from amicroscopic stage, as the initial stage, it becomes macroscopic, andthen soon reaches the optimum adaptation to the environment.Thus, while the damage suffered by paintings appears small in theearly stages, it is already well established when the first signs ofalterations become visible. Although a high number of fungalspores can contribute to modifications and alterations to paintingsin underground environments, these types of studies are very rare,and actually risk thresholds linked to biodeteriogens concentrationare not fixed and recognized commonly. Various aspects of hygieneare rather considered before conservation. In fact, high fungal andbacterial concentrations can be considered as indicator of un-healthy environment. Some health organizations set bacterial750 CFUm�3 and fungal 150 CFUm�3 as limit values (OccupationalSafety and Health Administration, United States Department ofLabour).

The literature gives numerous examples of allergic manifesta-tions after fungal contact. Most of these fungi are classified underAscomycetes and Deuteromycetes with a few in Basidiomycetes.

Many fungal genera considered in the present work are asso-ciated with symptoms of respiratory tract allergy. The allergenicand pathogenic potential of a range of fungi is well established, forexample, spores of Alternaria, Aspergillus, Penicillium and Clado-sporium spp. are responsible for causing an array of respiratoryconditions, from allergic rhinitis to asthma (Kurup et al., 2000).

It is important to remark that high concentrations of fungalspores in the air are not always index of risk for art-works becausenot all airborne species have biodeteriogen potential; it would betherefore necessary to know the relative concentration of thevarious species identifying those able to damage the materialsconserved in the analysed spaces.

Taking into account the objectives of the present study, whichinclude the characterisation of the indoor air quality, and theconservation and exhibition of the crypt, it was consideredappropriate to analyse all of the genera identified. This is alsobecause, as reported in the literature, these can be considered aspotential biodeteriogens of inorganic materials in undergroundenvironments (Monte and Ferrari, 1993; Nugari et al., 1993;Albertano and Urzì, 1999; Saarela et al., 2004; Cataldo et al.,2005; Sterflinger, 2010; Sterflinger and Pi~nar, 2013). An examplecan be seenwith underground frescos, which are often damaged bythe development of heterotrophic bacteria and fungi. Here, weshow that the most common fungal spores found in these confinedair spaces, such as in churches, chapels, crypts, and other similarclosed areas, are represented by the genera Cladosporium, Penicil-lium, Aspergillus, Alternaria and Fusarium. The comparison withother studies in Literature evidenced as the main genera recorded(Cladosporium, Penicillium, Aspergillus) in the present work were inline with those previously carried out (Montacutelli et al., 2000;Aira et al., 2007; Pangallo et al., 2012; Rojas et al., 2012). Thesetypes of spores can then also lead to serious consequences forplasterwork and paintings (Nugari et al., 1993). In particular on themural paintings, damages related to the colonization of micro fungibelonging to the family of Dematiaceae as Cladosporium, Stachybo-trys and Alternaria, manifested as brown stains, are frequent.Moreover, other species such as Trichoderma, Acremonium, Asper-gillus, Penicillium, Curvularia and Fusarium (Pietrini et al., 2005)

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130128

have been recorded. Different species of the genera Acremonium,Aspergillus, Chrysosporium, Cladosporium, Penicillium and Scopular-iopsis were already mentioned as inhabitants of indoor muralpaintings environments (Guglieminetti et al., 1994; Garg et al.,1995; Berner et al., 1997; Gorbushina and Petersen, 2000; Saarelaet al., 2004). It has been demonstrated that fungal growth in-duces serious deterioration, varying from superficial to profoundalterations of the painting structure, such as variously colouredstains, alteration of pigments and detachment of fragments byhyphal penetration considering that some fungal species can enterup to 10 mm inside the plaster (Nugari et al., 1998; Sterflinger,2010).

Among the strains isolated in Crypt of Otranto, Italy, there aredematiaceous such as Phoma and Alternaria. This “black fungi”, arethe most conspicuous and probably the most damaging organismsattacking and even penetrating the surfaces of stone monuments(Diakumaku et al., 1995; Urzì et al., 2000; Cataldo et al., 2005;Cappitelli et al., 2007:; Sterflinger and Pi~nar, 2013; Onofri et al.,2014; De Leo and Urzì, 2014).

The Trichoderma species, the main causes of deterioration ofmonuments have been often isolated in many German sandstonemonuments (Petersen et al., 1988; Saiz-Jimenez, 1995) and fromweathered sandstone in the Cathedral of Salamanca, Spain, in as-sociation with algae (De la Torre et al., 1991).

Furthermore, the ability of fungi to attack a wide range ofpolymeric substances, including those added to stone for protectivereasons, means that their presence has to be considered duringconservation treatments (Koestler and Santoro,1988; Salvadori andNugari, 1988; Leznicka et al., 1991; Koestler, 2000; Tiano et al.,2000; Lugauskas, 2003).

Polyurethane-degrading microorganisms including Fusariumsolani, Curvularia senegalensis, Aureobasidium pullulans and Clado-sporidium spp. were isolated (Crabbe et al., 1994) and esterase ac-tivity was detected with Curvularia senegalensis (Gu, 2003).

Species of Aspergillus, Cladosporium and Chaetomium are able todegrade polymeric substances and are also capable of growing onthe graphite (Gu, 2003). In a recent study (Pangallo et al., 2014)several fungal and bacterial strains were isolated from epoxy resins,which are frequently employed as consolidants of stone materials.The ability of Rhizopus microsporus and Aspergillus fumigatus togrow on an epoxy surface was also shown here for the first time,demonstrating that they have a role in epoxy deterioration.

Conclusions

Although the present study should be considered as a pre-liminary step in the investigation of the study area considered,important technical and methodological indications have beenobtained with a view to their further implementation in futureassessments.

The volumetric sampling allows to formulate standardized datathrough the aspiration of nominal air volumes, minimizing also thedifferences of airborne particles distribution potentially due todifferent air flows, temperatures and other environmentalvariables.

The Personal Volumetric Air Sampler spore trap for glass slidespermits to have a preliminary view of the airborne environment, arapid and exact knowledge of the environmental biologicalcontamination and therefore an information of the potential risksfor both the historical art objects and the operators of the field.Moreover, the volumetric sampling utilizing also the Andersensampler could furnish data on the airborne viable fungal particles.

Such environmental measures can provide valuable and crucialsupport to those who are responsible for sites of historical, artisticand archaeological heritage, for the identification of the most

suitable environments for temporary and permanent displays. Pe-riodic monitoring for the presence of biological aerosols, forexample, will allow the activation of procedures for risk preventionagainst fungal attack of sensitive materials. Early detection of thepresence of fungal spores or moulds where these artefacts arestored will allow, on the one hand, early intervention against mi-croorganisms that are potentially biodeteriogens, and, on the otherhand, the identification of micro-environments in which the cli-matic parameters are not suitable for conservation.

The definition of general guidelines for various structural andarchitectural environments can be quite complex, so it's essential,on the basis of general criteria, to identify the different factors thatcan lead to artefacts biological attacks to define properly thosefactors on which action can be taken. Among the prevention stra-tegies may be important to control any measures that may increasetemperature, relative humidity and carbon dioxide, factors whichcan cause the onset of biological attacks by different groups oforganisms.

The complete knowledge of the dynamics of the spore devel-opment in a specific site would permit to optimize the correctmanagement of the conservation environment and the most eco-nomic management of historical sites in need of restoration.

The information obtainedmay be important for future studies asthe control methods should be developed on a combination of in-formation about the characteristics of both the artefact materialsand the polymers and considering the microbial species.

The conservation intervention has to evaluate the consequenceson bio-susceptibility of the object potentially caused by the sameconservative method or by the materials utilized in restoration.

The detection of airborne fungal spores is of considerableimportance in this type of studies, as their degrading action playsan important role on the decay of both organic (wood, paper,leather) and inorganic materials (stone, marble, glass, metal),causing important changes not only from the aesthetic point ofview, but also structural, with loss of cohesion and solidity.

The aerobiological monitoring of fungal spores can provideessential information for appropriate interventions of preventionand restoration of cultural heritage and to assure it an adequateconservation in the “indoor” environments.

In summary the particular combination of the airborne fungalspores with the microclimatic conditions monitored in the studyarea, during specific periods of the year, could promote biodeteri-oration processes, determining a potential situation of risk for theCrypt. This condition suggest therefore the need to perform peri-odic monitoring for airborne pollution also according to the tem-perature and humidity trends.

Acknowledgements

The Authors would like to thank the Foundation for AgriculturalEducation in Perugia for giving us the permission to make aero-biological monitoring inside the crypt of the Basilica of St. Peter inPerugia.

Moreover, the Authors would like to thank Prof. Ulderico San-tamaria (Tuscia University, Italy) Director of the Vatican MuseumsDiagnostic Laboratory for Conservation and Restoration, for thehelpful comments and suggestions.

References

Aira, M.J., Jato, V., Stchigel, A.M., Rodrıguez-Rajo, F.J., Piontelli, E., 2007. Aero-mycological study in the cathedral of Santiago de Compostela (Spain). Int.Biodeterior. Biodegr. 60, 231e237.

Albertano, P., Urzì, C., 1999. Structural interactions among epilithic cyanobacteriaand heterotrophic microorganisms in roman hypogea. Microb. Ecol. 38,244e252.

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130 129

Angelosante Bruno, A., Pace, L., Tomassetti, B., Coppola, E., Verdecchia, M.,Pacioni, G., Visconti, G., 2007. Estimation of fungal spore concentrations asso-ciated to meteorological variables. Aerobiologia 23, 221e228.

Beech, I.B., 2004. Corrosion of technical materials in the presence of bio-filmsdcurrent understanding and state-of-the art methods of study. Int. Bio-deterior. Biodegrad. 53, 177e183.

Berner, M., Wanner, G., Lubitz, W., 1997. A comparative study of the fungal florapresent in medieval wall paintings in the chapel of the castle Herberstein and inthe parish church of St. Georgen in Styria, Austria. Int. Biodeterior. Biodegr. 40,53e61.

Booth, C., 1971. Methods in Microbiology, vol. 4. Academic Press, London and NewYork.

Camuffo, D., Bernardi, A., 1985. Fattori microclimatici e conservazione dei beniartistici. Edizioni del Laboratorio, Brescia.

Caneva, G., Nugari, M.P., Salvadori, O., 2002. La Biologia Nel Restauro. Nardini Edi-tore, Firenze.

Cappitelli, F., Zanardini, E., Sorlini, C., 2004. The biodeterioration of synthetic resinsused in conservation. Macromol. Biosci. 4, 399e406.

Cappitelli, F., Nosanchuk, J.D., Casadevall, A., Toniolo, L., Brusetti, L., Florio, S.,Principi, P., Borin, S., Sorlini, C., 2007. Synthetic consolidants attacked bymelanin-producing fungi: case study of the biodeterioration of Milan (Italy)cathedral Marble treated with acrylics. Appl. Environ. Microbiol. 271e277.

Cataldo, R., De Donno, A., De Nunzio, G., Leucci, G., Nuzzo, L., Siviero, S., 2005. In-tegrated methods for analysis of deterioration of cultural heritage: the crypt of“Cattedrale di Otranto”. J. Cult. Herit. 6, 29e38.

Chen, Y.P., Cui, Y., Dong, J.G., 2010. Variation of airborne bacteria and fungi at em-peror Qin's Terra-Cotta Museum, Xi’an, China, during the “Oct. 1” gold weekperiod of 2006. Environ. Sci. Pollut. Res. Int. 17, 478e485.

Ciferri, O., 1999. Microbial degradation of paintings. Appl. Environ. Microbiol. 65,879e885.

Crabbe, J.R., Campbell, J.R., Thompson, L., Walz, S.L., Scultz, W.W., 1994. Biodegra-dation of a colloidal ester-based polyurethane by soil fungi. Int. Biodeterior.Biodegrad. 33, 103e113.

De la Torre, M.A., Gomez-Alarcon, G., Melgarejo, P., Saiz-Jimenez, C., 1991. Fungi inweathered sandstone from Salamanca cathedral, Spain. Sci. Total Environ. 107,159e168.

De Leo, F., Urzì, C., 2014. Microfungi from deteriorated materials of cultural heritage.In: Misra, J.K., Tewari, J.P., Deshmukh, S.K., V�agv€olgyi, Csaba (Eds.), Fungi fromDifferent Substrates. CRC Press, Taylor & Francis Group, New York, pp. 144e158.A Science Publishers Book.

Diakumaku, E., Gorbushina, A.A., Krumbein, W.E., Panina, L., Soukharjevski, S., 1995.Black fungi in marble and limestonesdan aesthetical, chemical and physicalproblem for the conservation of monuments. Sci. Total Environ. 167, 295e304.

Garg, K.L., Jain, Kamal K., Mishra, A.K., 1995. Role of fungi in the deterioration of wallpaintings. Sci. Total Environ. 167, 255e271.

Gauzere, C., Moletta-Denat, M., Blanquart, H., Ferreira, S., Moularat, S., Godon, J.-J.,Robine, E., 2014. Stability of airborne microbes in the Louvre Museum overtime. Indoor air 24, 29e40.

Getty Conservation Institute, 1994. Preventive Conservation. In: Knell, S. (Ed.), Careof Collections. Routledge, London and New York, pp. 83e87.

Giacobini, C., Pietrini, A.M., Ricci, S., Roccardi, A., 1987. Problemi di bio-deterioramento. In: Bureca, A., Laurenzi Tabasso, M., Calandri, G. (Eds.), Mate-riali lapidei, problemi relativi allo studio del degrado e della conservazione, Vol.I supplemento al n. 41 del Bollettino d'arte. Istituto Poligrafico e Zecca delloStato, Roma, pp. 53e64.

Gorbushina, A.A., Petersen, K., 2000. Distribution of micro-organisms on ancientwall paintings as related to associated faunal elements. Int. Biodeterior. Biodegr.46, 277e284.

Gorbushina, A.A., Heyrman, J., Dornieden, T., Gonzalez-Delvalle, M., Krumbein, W.E.,Laiz, L., Petersen, K., Saiz-Jimenez, S., Swings, J., 2004. Bacterial and fungal di-versity and biodeterioration problems in mural painting environments of St.Martins church (GreeneeKreiensen, Germany). Int. Biodeterior. Biodegrad. 53,13e24.

Gravesen, S., 1979. Fungi as a cause of allergic disease. Allergy 34, 133e154.Gu, J.-D., 2003. Microbiological deterioration and degradation of synthetic poly-

meric materials: recent research advances. Int. Biodeterior. Biodegrad. 52,69e91.

Guglieminetti, M., De Guili Morghen, C., Radaelli, A., Bistoni, F., Carruba, G.,Spera, G., Caretta, G., 1994. Mycological and ultrastructural studies to evaluatebiodeterioration of mural paintings. Detection of fungi and mites in frescos ofthe monastery of St. Damian in Assisi. Int. Biodeter. Biodegr. 34, 269e283.

Hirsch, P., Eckhardt, F.E.W., Palmer, R.J., 1995. Fungi active in weathering of rock andstone monuments. Can. J. Bot. 73, 1384e1390.

Hirst, J.M., 1952. An automatic volumetric spore trap. Ann. Appl. Biol. 39, 257e265.Koestler, R.J., 2000. Polymers and resins as food for microbes. In: Ciferri, O., Tiano, P.,

Mastromei, G. (Eds.), Of Microbes and Art: the Role of Microbial Communities inthe Degradation and Protection of Cultural Heritage. Kluwer Academic/PlenumPublishers, New York, pp. 153e168.

Koestler, R.J., Santoro, E.D., 1988. Assessment of the Susceptibility to Biodeteriora-tion of Selected Polymers and Resins. GCI Scientific Program Report. The GettyConservation Institute, New York, p. 98 pp.

Kurup, V.P., Shen, H.-D., Banerjee, B., 2000. Respiratory fungal allergy. MicrobesInfect. 2, 1101e1110.

Leznicka, St, Kuroczkin, J., Krumbein, W.E., Strzelczyk, A.B., Petersen, K., 1991.Studies on the growth of selected fungal stains on limestones impregnated

with silicone resins (Steinfestiger H and Elastosil E-41). Int. Biodeterior. 28,91e111.

Lopez-Miras, M., Pi~nar, G., Romero-Noguera, J., Bolivar-Galiano, F.C., Ettenauer, J.,Sterflinger, K., Martin-Sanchez, I., 2013. Microbial communities adhering to theobverse and reverse sides of an oil painting on canvas: identification andevaluation of their biodegradative potential. Aerobiologia 29, 301e314. http://dx.doi.org/10.1007/s10453-012-9281-z.

Lugauskas, A., Levinskait�e, L., Peciulyt�e, D., 2003. Micromycetes as deteriorationagents of polymeric materials. Int. Biodeterior. Biodegrad. 52, 233e242.

Mandrioli, P., Caneva, G., 1998. Aerobiologia e beni culturali, Metodologie e tecnichedi misura. Nardini Editore, Firenze.

Mandrioli, P., Pasquariello, G., Roccardi, A., 2007. La prevenzione del bio-deterioramento. In: Caneva, G., Nugari, M.P., Salvadori, O. (Eds.), La Biologiavegetale per i beni culturali, Biodeterioramento e Conservazione, Vol. I. NardiniEditore, Firenze, pp. 273e307.

Milanesi, C., Baldi, F., Vignani, R., Ciampolini, F., Faleri, C., Cresti, M., 2006. Fungaldeterioration of medieval wall fresco determined by analysing small fragmentscontaining copper. Int. Biodeterior. Biodegrad. 57, 7e13.

Montacutelli, R., Maggi, O., Tarsitani, G., Gabrielli, N., 2000. Aerobiolgical moni-toring of the “Sistine Chapel”: airborne bacteria and microfungi trends. Aero-biologia 16, 441e448. http://dx.doi.org/10.1023/A:1026525432412.

Monte, M., Ferrari, R., 1993. Biodeterioration in subterranean environments. Aero-biologia 9, 141e148.

Nugari, M.P., Realini, M., Roccardi, A., 1993. Contamination of mural paintings byindoor airborne fungal spores. Aerobiologia 9, 131e139.

Nugari, M.P., Ricci, S., Roccardi, A., Monte, M., 1998. Chiese e Ipogei. In: Mandrioli, P.,Caneva, G. (Eds.), Aerobiologia e beni culturali. Metodologie e tecniche dimisura. Nardini Editore, Firenze, pp. 229e246.

Nugari, M.P., Roccardi, A., 2001. Aerobiological investigations applied to the con-servation of cultural heritage. Aerobiologia 17, 215e223.

Nugari, M.P., 2003. The aerobiology applied to the conservation of works of art.Coalition 6, 8e10.

Nugari, M.P., Roccardi, A., Cacace, C., 2007. La Cripta di San Magno ad Anagni:indagini aerobiologiche e microclimatiche. In: Bollettino Istituto Centrale per ilRestauro Nuova Serie, vol. 14, pp. 76e80.

Ogden, E.C., Raynor, G.S., Hayes, G.V., Lewis, D.M., Haines, J.H., 1974. Manual forSampling Airborne Pollen. Hafner Press, NY.

Onofri, S., Zucconi, L., Isola, D., Selbmann, L., 2014. Rock-inhabiting fungi and theirrole in deterioration of stone monuments in the Mediterranean area. PlantBiosyst. 148, 384e391.

Orlandi, F., Bonofiglio, T., Sgromo, C., Ruga, L., Romano, B., Fornaciari, M., 2011. Anapplied aerobiological study to test the efficacy of pollen filters in limiting in-door pollen contamination. Grana 50, 73e80.

Palombaro, F., 2007. Architettura Nel Primo Millennio Cristiano a Perugia. Quat-troemme Editore, Perugia.

Pangallo, D., Krakov�a, L., Chovanov�a, K., Simonovicov�a, A., De Leo, F., Urzì, C., 2012.Analysis and comparison of the microflora isolated from fresco surface andfrom surrounding air environment through molecular and biodegradative as-says. World J. Microbiol. Biotechnol. 28, 2015e2027. http://dx.doi.org/10.1007/s11274-012-1004-7.

Pangallo, D., Bu�ckov�a, M., Krakov�a, L., Pu�sk�arov�a, A., �Sakov�a, N., Grivalský, T.,Chovanov�a, K., Zem�ankov�a, M., 2014. Biodeterioration of epoxy resin: a micro-bial survey through culture-independent and culture-dependent approaches.Environ. Microbiol. http://dx.doi.org/10.1111/1462-2920.12523.

Pasquarella, C., Sansebastiano, G.E., Saccani, E., Ugolotti, M., Mariotti, F., Boccuni, C.,Signorelli, C., Fornari Schianchi, L., Alessandrini, C., Albertini, R., 2011. Proposalfor an integrated approach to microbial environmental monitoring in culturalheritage: experience at the Correggio exhibition in Parma. Aerobiologia 27,203e211. http://dx.doi.org/10.1007/s10453-010-9189-4.

Pelizzari, F., 1996. Gravimetric survey of airborne fungal spores in Milan. Aero-biologia 12, 205e207.

Petersen, K., Kuroczkin, J., Strzelezyk, A.B., Krumbein, W.E., 1988. Distribution andeffects of fungi on and in sandstones. In: Houghton, D.R., Smith, R.N.,Eggins, H.O.W. (Eds.), Biodeteration 7. Elsevier Applied Science, London,pp. 455e460.

Pietrini, A.M., Ricci, S., Nugari, M.P., 2005. Chiese e cripte. In: Caneva, G.,Nugari, M.P., Salvadori, O. (Eds.), La Biologia vegetale per i beni culturali,Biodeterioramento e Conservazione, vol. I. Nardini Editore, Firenze,pp. 179e188.

Pinna, D., 2003. The prevention of biodeterioration. Coalit. Newsl. 6, 14e16.February.

Pyrri, I., Kapsanaki-Gotsi, E., 2007. A comparative study on the airborne fungi inAthens, Greece, by viable and non-viable sampling methods. Aerobiologia 23,3e15. http://dx.doi.org/10.1007/s10453-006-9039-6.

Qian, J., Hospodsky, D., Yamamoto, N., Nazaroff, W.W., Peccia, J., 2012. Size-resolvedemission rates of airborne bacteria and fungi in an occupied classroom. IndoorAir 22, 339e351.

Rivas-Martinez, S., 1996. Clasificacion bioclimatica dela tierra. Folia Bot. Matr. 16,1e32.

Rojas, T.I., Aira, M.J., Batista, A., Cruz, I.L., Gonz�alez, S., 2012. Fungal biodeteriorationin historic buildings of Havana (Cuba). Grana 51, 44e51.

Ruga, L., Bonofiglio, T., Sgromo, C., Orlandi, F., Romano, B., Fornaciari, M., 2007.Presenza di biodeteriogeni nel percorso museale “Il Perugino” e possibili rela-zioni con l'indoor e l'outdoor. In: Bollettino Istituto Centrale per il RestauroNuova Serie, vol. 14, pp. 66e70.

L. Ruga et al. / International Biodeterioration & Biodegradation 98 (2015) 121e130130

Ruga, L., Bonofiglio, T., Orlandi, F., Romano, B., Fornaciari, M., 2008. Analysis of thepotential fungal biodeteriogen effects in the “Doctorate Library” of the uni-versity of Perugia, Italy. Grana 47, 60e69.

Saarela, M., Alakomi, H.L., Suihko, M.L., Maunuksela, L., Raaska, L., Mattila-Sandholm, T., 2004. Heterotrophic microorganisms in air and biofilm samplesfrom roman catacombs, with special emphasis on actinobacteria and fungi. Int.Biodeterior. Biodegrad. 54, 27e37.

S�aenz Laín, C., Monserrat Guti�errez Bustillo, A., 2006. In: Esporas atmosf�ericas en laComunidad de Madrid. Instituto de Salud Pública, Madrid.

Saiz-Jimenez, C., 1995. Deposition of anthropogenic compounds on monuments andtheir effect on airborne microorganisms. Aerobiologia 11, 161e175.

Salvadori, O., Nugari, M.P., 1988. The effect of microbial growth on synthetic poly-mers used onworks of art. In: Houghton, D.R., Smith, R.N., Eggins, H.O.W. (Eds.),Biodeterioration, vol. 7. Elsevier Applied Science, London, New York,pp. 424e427.

Sorlini, C., 1993. Aerobiology: general and applied aspects in the conservation of artworks. Aerobiologia 9, 109e115.

Sterflinger, K., 2010. Fungi: their role in deterioration of cultural heritage. FungalBiol. Rev. 24, 47e55.

Sterflinger, K., Pi~nar, G., 2013. Microbial deterioration of cultural heritage and worksof art d tilting at windmills? Appl. Microbiol. Biotechnol. 97, 9637e9646.http://dx.doi.org/10.1007/s00253-013-5283-1.

Tiano, P., Biagiotti, L., Bracci, S., 2000. Biodegradability of products used inmonuments' conservation. In: Ciferri, O., Tiano, P., Mastromei, G. (Eds.), Of

Microbes and Art: the Role of Microbial Communities in the Degradation andProtection of Cultural Heritage. Kluwer Academic Publishers, Dordrecht,pp. 169e182.

Urzì, C., De Leo, F., Hoog de, G.S., Sterflinger, K., 2000. Recent advances in the mo-lecular biology and ecophysiology of meristematic stone-inhabiting fungi. In:Ciferri, O., Tiano, P., Mastromei, G. (Eds.), Of Microbes and Art. The Role ofMicrobial Communities in the Degradation and Protection of Cultural Heritage.Kluwer Academic/Plenum, New York, pp. 3e19.

Urzì, C., De Leo, F., Salamone, P., Criseo, G., 2001. Airborne fungal spores colonisingmarbles exposed in the terrace of Messina Museum, Sicily. Aerobiologia 17,11e17.

Warscheid, Th, Braams, J., 2000. Biodeterioration of stone: a review. Int. Biodeterior.Biodegrad. 46, 343e368.

Wollenzien, U., de Hoog, G.S., Krumbein, W.E., Urzì, C., 1995. On the isolation ofmicrocolonial fungi occurring on and in marble and other calcareous rocks. Sci.Total Environ. 167, 287e294.

Zammit, G., De Leo, F., Urzì, C., Albertano, P., 2009. A non-invasive approach to thepolyphasic study of biodeteriogenic biofilm at St Agatha Crypt and Catacombsat Rabat, Malta. In: “Science and Cultural Heritage in the Mediterranean Area eDiagnostics, Conservation Experiences and Proposals for a Risk Map” Confer-ence Proceedings, Palermo 2007, vol. 15. Quaderni Palazzo Montaldo, Italy,pp. 323e327.