International Biodeterioration & Biodegradation 157 (2021) 105163

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International Biodeterioration & Biodegradation

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Diversity and seasonal dynamics of culturable airborne fungi in a cultural

heritage conservation facility
Željko Savković a, *, Miloš Stupar a, Nikola Unković a, Žarko Ivanović b, Jovana Blagojević b,
Slađana Popović c, Jelena Vukojević a, Milica Ljaljević Grbić a
a
University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden ‘‘Jevremovac’‘, Takovska 43, 11000, Belgrade, Serbia
b
The Institute for Plant Protection and Environment, Teodora Drajzera 9, 11040, Belgrade, Serbia
c
University of Belgrade, Scientific Institution, Institute of Chemistry, Technology and Metallurgy, National Institute, Department of Ecology and Technoeconomics,
Njegoševa 12, 11000, Belgrade, Serbia

A R T I C L E I N F O A B S T R A C T

Keywords: An extensive aeromycological survey was performed in the premises of a cultural heritage conservation facility to
Airborne fungi estimate fungal propagule concentrations in both indoor and outdoor air and seasonal dynamics with regard to
Aspergillus temperature and air humidity. The highest average propagule concentration in indoor air was documented in
Cladosporium
spring, and for outdoor air, in summer. Increased propagule loads were documented in most rooms during
Cultural heritage
Human pathogens
winter, fall and spring, and in all examined rooms in summer. Very high propagule concentrations (>25,000
Penicillium CFU m− 3) were reported in ground-floor rooms. The total mycobiota was comprised of 74 fungal species, with a
prevalence of Aspergilli and Penicillia. Cladosporium and Penicillium isolates were found to be the most abundant in
all seasons in both indoor and outdoor air samples. Among the identified fungi, potential human pathogens,
allergens and mycotoxin producers were present. Numerous documented species play an important role in the
deterioration of cultural heritage artifacts and are known producers of extracellular enzymes, acids and pig­
ments. Application of a selective medium (M40Y) allowed isolation of various xerophiles and xerotolerant
species. Aeromycological analyses are mandatory for determining the appropriate conditions for the protection
of the health of conservators, as well as the objects of cultural heritage.

1. Introduction
Indoor cultural heritage premises, such as museums, libraries, ar­
chives and storage depositories, are frequently located in old or monu­
ment buildings. However, only a small number of these can be
considered properly constructed and maintained (Ferdyn-Grygierek
et al., 2020). In such environments, various factors may cause significant
temperature and humidity variations, which can elevate bioaerosol
concentrations, increasing the risks to stored cultural heritage objects
and human health (Frankel et al., 2012; Awad et al., 2020). In recent
years, there has been a rising trend in aerobiological studies of the risks
to cultural heritage artifacts in buildings where they are kept (Niesler
et al., 2010; Harkawy et al., 2011; Nunes et al., 2013; Borrego and
Perdomo, 2016). Moreover, multidisciplinary studies on cultural heri­
tage environment have been performed including biological monitoring,
microclimate analysis linked to Computational Fluid Dynamic (CFD), in
order to define standardized protocols (Pasquarella et al., 2015).
Fungal propagules, mainly spores and mycelial fragments, are al­
ways present in indoor and outdoor air, their concentrations being
dependent on environmental factors (Kasprzyk, 2008). Fungi produce
various types of sexual and asexual spores during their life cycle. Spores
are then actively or passively released into the surrounding air and
dispersed by air currents to available substrates. Dispersion of both
viable and nonviable fungal structures can occur over long distances and
depends not only on their dimensions, but also on their biological fea­
tures and different environmental factors: air temperature, oxygen
availability, nutrients presence, the texture and vibrations of the landing
surfaces (Hjelmroos, 1993; Viegas et al., 2015). The successful coloni­
zation of available substrata requires viable propagules and favorable
growth conditions (humidity, temperature, nutrients) (Florian, 2002;
Kasprzyk, 2008).
The presence and concentration of airborne fungal spores in indoor
spaces are linked to various types of automated and human activities
such as air-conditioning systems, ventilation, hygiene and others

* Corresponding author.
E-mail addresses: zsavkovic@bio.bg.ac.rs (Ž. Savković), smilos@bio.bg.ac.rs (M. Stupar), unkovicn@bio.bg.ac.rs (N. Unković).

https://doi.org/10.1016/j.ibiod.2020.105163
Received 31 January 2020; Received in revised form 13 December 2020; Accepted 14 December 2020
Available online 17 December 2020
0964-8305/© 2020 Elsevier Ltd. All rights reserved.
Ž. Savković et al.
(Samson et al., 2010). Contaminated cultural heritage artifacts could
present an additional source of fungal contamination of conservation
facilities, especially if not handled in the proper manner (Sterflinger,
2010). In the abovementioned premises, aerobiological investigations
are essential for detecting sources of contamination, to assess flow and
areas of major accumulation of the airborne fungi, as well as to deter­
mine diurnal and seasonal variations (Nugari and Roccardi, 2001; Bor­
rego and Perdomo, 2016). Monitoring of fungal bioaerosols provides
information concerning the hazards of contamination of stored cultural
heritage objects and, more importantly, enables evaluation the potential
health impact (Florian, 2002). Airborne propagules of allergenic, toxi­
genic and pathogenic fungi are often present in indoor spaces (Unković
et al., 2018a). It is therefore essential to monitor the quantitative and
qualitative structure of fungal bioaerosols regularly, especially if po­
tential novel sources of contamination appear. The aim of this research
was to determine the seasonal quantitative and qualitative fungal bio­
aerosol composition in a facility for the conservation of cultural heritage
objects.
2. Materials and methods
2.1. Study sites
The study was carried out in the premises of the Central Institute for
Conservation in Belgrade, Serbia. An aeromycological survey was per­
formed during the four seasons of 2014/2015 period. Measurements
were done in 20 rooms of different purpose and in immediate proximity
to the building, in front of the entrances.
Sampling sites included: a) the ground floor: ateliers for the con­
servation of stone artifacts (I1, I2, I3 and I4), storage room – out of use
(I5), hallway (I6); b) the first floor: depot (I7), ateliers for the conser­
vation of paintings (I8, I9 and I10), for the conservation of textile (I11,
I12), and for the conservation of metal artifacts (I13), quarantine room
(I14), hallway (I15); c) the second floor: photo studio (I16), hallway
(I17); d) the third floor: atelier for the conservation of ceramics (I18),
library (I19), hallway (I20); and e) outdoor sampling sites (O1 – in the
courtyard near the main entrance, O2 – in proximity of the back
entrance).
2.2. Measurement of microclimate parameters
Temperature (T, ◦ C) and relative air humidity (RH, %) were
measured using a Temperature and Humidity Data Logger with a probe
(TESTO 635) in each investigated room and outdoors during all four
seasons.
2.3. Air sampling and estimation of propagule concentrations
Fungal propagules were collected by the volumetric air-sampling
method. An air volume (100 L) was vacuumed using a MAS-100 Eco
Air Sampler and subsequently inoculated on three different mycological
media: Malt Extract Agar (MEA) and Oatmeal Agar (OA) for mesophilic
fungi, Malt Yeast 40% sucrose extract (M40Y) for xerophiles and xero­
tolerant species. Petri dishes with inoculated media were transported to
the laboratory and incubated in a thermostat (UE 500, Memmert) at 25
± 2 ◦ C for 7 days. After the incubation period, the Petri dishes were
examined, and all visible colonies were counted and labeled. The ob­
tained numbers of the colonies were then converted to probable statis­
tical total values, calculated from formula 1 (Feller, 1950):
Pr = N [1/N + 1/(N-1) + 1/(N-2) + … + 1/(N-r+1)] [1]
Pr – probable statistical total, N – number of holes in perforated plate
(400), r – number of colonies formed on medium in Petri dish (Ø 90
mm).
And expressed as CFU m− 3 units for each room and outdoor site. The
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International Biodeterioration & Biodegradation 157 (2021) 105163
total propagule load for indoor air was expressed as a mean value with
standard error for each season and mycological medium, separately.
The relative frequency (RF) of specific fungal taxa was determined
according to Esquivel et al. (2003):
number ​ of ​ times ​ a ​ genus ​ is ​ detected
RF ​ (%) = × 100 [2]
total ​ number ​ of ​ samplings ​ realized
The obtained RF values were used to categorize the detected fungal
genera into several groups as follows: abundant (81–100%), common
(61–80%), frequent (41–60%), occasional (21–40%), and rare
(0.1–20%) (Borrego and Perdomo 2016).
The relative density (RD) of specific fungal taxa was calculated as per
the formula of Smith (1980):
number ​ of ​ colonies ​ of ​ the ​ genus ​ or ​ species
RD ​ (%) = × 100
total ​ number ​ of ​ colonies ​ of ​ all ​ genera ​ or ​ species
[3]
2.4. Fungal identification
Morphologically different colonies were examined and identified
based on colony morphology (observed with a stereomicroscope Stemi
DV4, Carl Zeiss) and the microscopic characteristics of reproductive
structures (using a light microscope Zeiss Axio Imager M.1 with Axio­
Vision release 4.6 software). Identification of fungi was performed using
the following identification keys: Ellis and Ellis (1997), Watanabe
(2002), Samson et al. (2010), Bensch et al. (2012) and Woudenberg et al.
(2013).
Identification of isolates was confirmed using molecular methods. A
total of 120 morphologically different fungal isolates were selected for
molecular characterization, reinoculated on MEA, M40Y and OA media
and incubated at 25 ± 2 ◦ C for 7 days. Dry marginal mycelia (40 mg)
were collected and used for DNA extraction according to the manufac­
turer’s instructions of a DNA Mini Kit (Qiagen, Valencia, CA, USA). PCR
amplification of the ITS region was conducted using the primers ITS1/
ITS4 (White et al., 1990), and Bt2a/Bt2b primers (Glass and Donaldson,
1995) were used for amplification of the β-tubulin gene. PCR amplifi­
cation of ITS regions was performed as follows: initial denaturation at
95 ◦ C for 4 min, 35 amplification cycles at 95 ◦ C for 30 s, 52 ◦ C for 1 min
and 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 7 min. PCR
amplification of β-tubulin was conducted as follows: initial denaturation
at 94 ◦ C for 4 min, 35 amplification cycles at 94 ◦ C for 1 min, 58 ◦ C for 1
min and 72 ◦ C for 1 min, with a final extension phase at 72 ◦ C for 7 min.
Amplification reactions were performed in a Mastercycler personal
model (Eppendorf, Hamburg, Germany) in a reaction mixture (25 μl),
using the following final concentrations or total amounts: 5 ng DNA, 1 ×
PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 μM of each primer,
2.5 mM MgCl2, 0.25 mM of each dNTP, 1 unit of Taq polymerase. The
amplified DNA fragments were fractionated in 1% agarose gels in 0.5 ×
TBE buffer, and visualized by ethidium bromide staining and UV illu­
mination. The resulting PCR products were separated by electrophoresis
and purified (Purification Kit, Qiagen, USA) for sequencing (Macrogene,
Seoul, South Korea). Sequences were compared with other related se­
quences from the NCBI database using the BLAST program (BLAST+
2.7.1 of the NCBI) for primary identification. Fungal sequences were
deposited in the NCBI GenBank (see Supplementary materials).
2.5. Statistical analyses
Three canonical correspondence analyses (CCA) were performed on
a set of data involving indoor fungal taxa where the number of isolates of
all recorded taxa was used as a measure. CCA in all analyses were used
since the gradient was 3.3 SD units long. The first CCA examined the
relationship between fungal taxa and type of nutrient media (MEA,
M40Y and OA) on which species were isolated, while the second CCA
focused on the relationship between fungal taxa and seasons (Spring,
Ž. Savković et al.
Summer, Fall and Winter). The third CCA was performed to observe
fungal composition in relation to T and RH (explanatory variables) and
CFU m− 3 and the rooms from which samples were taken I1–I20 (sup­
plementary variables).
3. Results
3.1. Microclimate parameters
Average temperatures measured indoors ranged from 22.44 ±
0.88 ◦ C to 26.21 ± 0.40 ◦ C throughout the study period (Fig. 1). Outdoor
average temperatures ranged from 7.15 ± 2.95 ◦ C to 30.15 ± 0.35 ◦ C.
Additionally, the relative air humidity in investigated rooms varied from
33.04 ± 2.47% to 54.92 ± 1.53%. Outdoor air humidity was in the range
of 38.05 ± 4.35% to 81 ± 5.5% (Fig. 1).
3.2. Air mycobiota
The maximal measured fungal propagule concentration (>25,000
CFU m− 3) was recorded in 6 rooms during spring, 2 rooms in winter and
1 room in the fall. These were: the ground-floor hallway (I6), storage
room (I5), ateliers for the conservation of stone artifacts (I1–I4), atelier
Fig. 1. Average spore loads in indoor air of the cultural heritage conservation
facility (a) and outdoor air of its immediate surroundings (b) sampled on three
microbiological media (MEA, M40Y and OA) in relation to temperature (T) and
relative air humidity (RH) data. Values in the left vertical axis are shown in
logarithmic scale.
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International Biodeterioration & Biodegradation 157 (2021) 105163
for the conservation of ceramics (I18) and the quarantine room (I14).
The highest average propagule concentration was documented on M40Y
in spring (7153 ± 2490 CFU m− 3) for indoor air and on MEA in summer
(5960 CFU m− 3) for outdoor air, while the lowest was documented in
winter on M40Y and OA, respectively, for both indoor and outdoor sites
(168.5 ± 38.30 CFU m− 3 and 540 CFU m− 3) (Fig. 1).
A total of 74 fungal species from 23 genera were identified from
indoor and outdoor samples (Supplementary Table 1). Most isolated
species belonged to the genus Aspergillus (19) followed by Penicillium
(15), Fusarium (8), Alternaria (6), Cladosporium (4), Talaromyces (3) and
Arthrinium (3). Other genera were represented with only one identified
species.
Based on RF values, the most prevalent indoor genera were Clado­
sporium and Penicillium. Isolates of these genera were estimated to be
abundant during spring, summer and fall but common during winter
(Fig. 2). RD values confirmed these genera as the most frequent.
(Table 1).
Concerning outdoor samples, RD values showed that Cladosporium
and Penicillium isolates were most numerous as well. In a seasonal
comparison, isolates of these genera were most frequent in summer and
least frequent in the fall (Table 1).
3.3. Statistical data
The first CCA (F = 5.6, P = 0.0002) was used to illustrate the pref­
erence of recorded taxa for a certain medium. The majority of recorded
taxa (placed in the center of the CCA ordination diagram) were isolated
from all three media. However, some of them still preferred one medium
more than the other two, for example, Trichoderma, Fusarium and
Arthrinium species were mostly isolated from OA and Aspergillus species
from M40Y. On the other hand, some representatives were isolated from
two or only one medium: Nothophoma and Sclerotinia species were iso­
lated from M40Y and OA, while Beauveria and Clonostachys species were
exclusively isolated from MEA (Fig. 3a).
The pie symbol diagram of the second CCA (F = 8.8, P = 0.0002)
illustrates the relationship of documented taxa and seasons that were
used as explanatory variable. Some taxa were recorded in only one
season (Beauveria in spring, Sclerotinia in winter and Clonostachys in
summer), while others were found in two seasons (Botrytis and Arthri­
nium were documented in spring and winter, and Nothophoma in fall and
spring). Those in the central part of the ordination diagram were found
in all seasons, with the majority predominantly registered in summer,
such as Cladosporium and Fusarium species. Conversely, Penicillium spe­
cies were mostly recorded during spring and Aspergillus species during
fall and summer.
The third CCA (F = 9.0, P = 0.0002) was performed to assess the
Fig. 2. Relative frequencies (RFs) of selected fungal genera in indoor air of the
cultural heritage conservation facility during the study period.
Ž. Savković et al. International Biodeterioration & Biodegradation 157 (2021) 105163

Table 1
Relative densities (RDs, %) for the most frequent fungal genera from indoor air in the cultural heritage conservation facility and in its immediate surroundings, during
the study period, on tested media.
Genera MEA M40Y OA

Fall Winter Spring Summer Fall Winter Spring Summer Fall Winter Spring Summer

Indoor air
Cladosporium 12.78 14.92 22.11 81.82 2.34 7.69 25.78 54.98 2.55 3.05 17.40 83.75
Penicillium 16.28 9.92 49.63 6.40 14.70 16.62 58.33 29.68 20.01 3.54 44.16 7.54
Aspergillus 2.93 0.59 0.08 2.44 47.77 11.08 4.40 8.42 – 0.19 0.08 1.23
Alternaria 0.05 0.42 0.53 1.43 – 5.54 5.70 0.64 0.09 0.23 0.45 2.23
Fusarium – 0.42 0.10 1.37 – – 0.06 0.18 0.14 0.12 0.72 2.49
Epicoccum 0.26 0.08 0.48 0.84 – 0.62 0.34 0.29 0.23 0.18 0.24 0.23
Trichoderma 0.31 – – 0.22 – – – – 0.23 0.06 – 0.71
Outdoor air
Cladosporium 3.50 26.79 82.79 89.18 2.36 62.71 38.61 65.95 8.26 22.77 33.05 91.24
Penicillium 6.99 3.92 1.21 7.84 0.79 2.82 7.91 8.11 9.09 15.84 3.67 3.85
Aspergillus 2.10 – – 0.19 33.86 1.69 3.80 7.75 – – – 1.50
Alternaria – 2.61 2.02 0.93 – 7.91 23.73 3.78 – 1.98 3.95 1.71
Epicoccum 2.10 0.65 1.01 0.37 – 2.82 2.22 1.26 – 2.97 1.41 0.21
Fusarium – – – – – – 0.32 – 1.65 – 1.41 1.07

potential influence of T and RH on fungal composition where CFU m− 3
and the rooms (I1–I20) from which samples were taken are included as
supplementary variables. The relationships of documented fungal taxa
and explanatory variables (T and RH) are presented in Fig. 3c, and the
arrangement of all explanatory and supplementary variables can be seen
in Fig. 3d. Both graphs are from the same CCA. As regards rooms
(Fig. 3d), two groups can be distinguished. The first group (I1–I6) in the
upper part of the ordination diagram refers to the rooms where a higher
RH (average RH was mostly above 45%) and lower T (average T mostly
below 24 ◦ C) were measured. The second group (shaded area in the
lower left part of the ordination diagram) includes rooms where a lower
RH (average RH mostly lower than 45%) and higher T (average T mostly
higher than 24 ◦ C) were measured. Many representatives were associ­
ated with this second group where a higher T was recorded. Many
recorded taxa were positively correlated to T (Fig. 3c), only a few
showed negative correlations, among which were Nothophoma, Scle­
rotinia and Basidiomycota. On the other hand, a few fungal taxa showed
a slightly positive correlation to RH (i.e. Aspergillus, Penicillium) while
others were negatively correlated (taxa from the lower part of the
ordination diagram, i.e. Sclerotinia, Arthrinium, Botrytis, etc.). However,
when considering CFU m− 3, higher values were recorded in the first
group where the RH was also higher, while a lower CFU m− 3 was
recorded in the second group.
4. Discussion
The study demonstrates viable fungal propagule concentrations from
a large number of samples in a cultural heritage conservation facility
with a seasonal distribution on different mycological media. The
investigated fungal propagule load and diversity of isolates are essential
determining potential threats to both cultural heritage materials and
human health.
Propagule concentrations in the facility’s rooms reached extremely
high values in all tested seasons, especially during spring and summer.
In fact, values higher than 150 CFU m− 3, according to standards for
Italian libraries (Micheluz et al., 2015), were documented in most rooms
during winter, fall and spring as well as in all examined rooms in sum­
mer. It should be noted that in some rooms, especially in the
ground-floor hallway and storage room, propagule concentrations were
even above 25,000 CFU m− 3. These data point ot a remarkably high
fungal contamination in the investigated indoor environment. However,
it should be noted that approximately only 1% of microorganisms pre­
sent in the environment can be cultivated, based on the observation that
microscopic counts are considerably larger than the equivalent total
viable counts (Ward et al., 1990; DeLong et al., 1994; Vartoukian et al.,
2010). This implies that propagule loads in the investigated premises
could be even higher than recorded and at the same time presents a main
issue when using standard isolation methods. To date, there is no official
reference limit for levels of fungal propagule concentration in indoor air
and quantitative standards range from less than 100 CFU m− 3 to greater
than 1000 CFU m− 3 of total fungal propagules (Rao et al., 1996). World
Health Organization (1990) standards proposed that up to 500 CFU m− 3
of fungi from indoor sources could be considered acceptable if the spe­
cies present were primarily Cladosporium and other common phyllo­
plane fungi, although more recent standards proposed by World Health
Organization experts stipulate a limit of 1000 CFU m− 3 (Nevalainen and
Morawaska, 2009). Concerning cultural heritage facilities, the recom­
mended limits are 1000 CFU m− 3 for archives (Borrego et al., 2010;
Nunes et al., 2013) and 150 CFU m− 3 for Italian libraries (Micheluz
et al., 2015).
Numerous factors, such as specific building material composition,
presence of water damage, condensation, type of ventilation system as
well as the presence of dust and food leftovers etc. affect indoor air spore
concentration and composition (Samson et al., 2010). Since indoor
temperatures usually do not fluctuate greatly and dust particles carry
different nutrients, it is thought that temperature and nutrient avail­
ability usually do not present a limiting factor for indoor fungal growth,
although they could affect growth rate, as well as fungal metabolism. On
the other hand, moisture is a critical factor for fungal growth and pro­
liferation, and elevated numbers of fungal species and spore emissions
are reported in damp indoor environments (World Health Organisation
Europe, 2009). It is well known that increased fungal growth and sub­
sequent abundant emissions of spores occur in archives, museums and
conservation facilities when the relative air humidity is high or water
accumulates due to leakages or condensation (Samson et al., 2010). Our
study shows increased fungal propagule loads in rooms with higher RH
values, specifically in the rooms on the ground floor (I1–I6) (Fig. 3d). It
is well known that thermohygric parameters significantly influence
microbial colonization on artworks (De Nuntiis and Palla, 2017; Awad
et al., 2020). However, it should be emphasized that although temper­
ature and RH could impact fungal propagule elevation in indoor air,
they cannot fully explain the observed seasonal distribution (Frankel
et al., 2012). Aside from RH, which was elevated during the study
period, it should be mentioned that the ground-floor premises also had
poor ventilation because they were enclosed, but they had a constant
influx of contaminated artifacts that are kept there awaiting restoration.
Additionally, the storage room (I5) located on the ground floor, has not
been used for a long period of time, and is occasionally flooded by un­
derground water. At the same time, it was also observed that elevated
fungal propagule concentrations were higher in some rooms of the
building’s upper floors, i.e. in places where different types of automated
and human activities are more frequent. As mentioned, the abundance of

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Ž. Savković et al. International Biodeterioration & Biodegradation 157 (2021) 105163

Fig. 3. CCA showing the relationships between: a) documented fungi and mycological media (MEA, M40Y and OA); b) documented fungi and seasons (Spring (red),
Summer (green), Fall (white) and Winter (gray)); c) and d) documented fungal taxa, explanatory variables (RH and T) and supplementary variables (CFU m− 3 and
sampling sites – rooms (I1–I20)). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

airborne fungal propagules in indoor environments depends on
anthropogenic factors such as air-conditioning systems, ventilation,
hygiene, etc. (Samson et al., 2010).
In our study, the isolates of some genera (e.g. Fusarium, Beauveria and
Trichoderma) were less numerous. It is known that the spores of these
species are kept aggregated by a slimy substance which makes them
heavy and difficult to be transported by air (Viegas et al., 2015). On the
contrary, Aspergillus, Penicillium and Cladosporium isolates were quite
common in our samples. The same species form dry walled spores, which
easily disassociate and are very light, rendering them very easily
dispersible (Viegas et al., 2015). Thus, dry spores can be quite numerous
in indoor air and can be easily inhaled, in contrast to wet spores, which
are formed in slimy heads (Samson et al., 2010; Viegas et al., 2015).
Finally, concentrations of indoor fungal spores are also significantly
dependent on outdoor air influx (Adams et al., 2013; Nevalainen et al.,
2015). This could be one of causes affecting spore concentration in the
facility’s indoor air, since higher indoor propagule concentrations were
measured during spring and summer periods and were the lowest in
winter, as they were outdoors.
In cases of high spore loads, it is also necessary to investigate the
possible sources of contamination. Since the ateliers of the facility serve
for the conservation and deposition of different artifacts, it is quite
possible that contamination was introduced from the outside. Objects
accepted for conservation treatment are from different backgrounds and
bring different mycobiota. Since during conservation treatment an ob­
ject moves through the building along a certain path, it was important to
undertake an aerobiological sampling that followed the movement of
objects: entrance, hallways, photo studio, atelier and storage. One spe­
cific example is an ancient Roman stela that was brought to the facility
for restoration with high degree of fungal contamination (Savković
et al., 2016). Furthermore, it should be noted that a several dozen of
people are present in the facility on the everyday basis (including con­
servators, trainees and other employees) while the number of visitors is
negligible. Therefore, the circulation of people could be one of the

5
Ž. Savković et al.
reasons for propagules transmission through the air within the premises.
However, this is less significant than the input of propagules brought in
by the infested artifacts themselves.
Concerning the outdoor air samples in this research, the average
propagule concentrations peaked in summer and were lowest in winter.
It was observed that in temperate climate conditions, the maximum
concentrations of most species’ spores occur in summer or early fall
(Kasprzyk and Worek, 2006; Kasprzyk, 2008). Conversely, during the
winter and early spring, when suitable substrates are scarce, the con­
centration of fungal spores is relatively low compared to warmer seasons
(Kasprzyk, 2008).
From a total of 120 morphologically different fungal isolates, 74
species were confirmed by molecular identification methods which
suggests high fungal diversity with the most frequently isolated species
predominantly belonging to ascomycetes (Aspergillus and Penicillium,
followed by Fusarium, Alternaria and Cladosporium species). It should be
noted that these genera are usually fast growing in culture and are also
ubiquitous, therefore, its presence is expected when standard culture
media are used. On the other hand, many plant pathogens (such as
Botrytis, Didymella, Microsphaeropsis, Nothophoma and Sclerotinia spe­
cies) and basidiomycetes (Bjerkandera, Coprinellus, Irpex, Schizophyllum
and Stereum) were represented by one species of each genera. Apart from
the high documented diversity of fungal taxa in our research, species of
the genera Cladosporium, Penicillium and Aspergillus were recorded as the
most abundant indoor airborne fungi of the facility. This confirms the
well-established opinion that these are the dominant fungal genera
recorded in indoor environments and corresponds with results reported
in other studies (Pei-Chih et al., 2000; Shelton et al., 2002; de Ana et al.,
2006). Based on RF and RD values, our results also suggest the preva­
lence of Cladosporium and Penicillium spores indoors during all seasons.
Cladosporium spores were most numerous in summer, while the spores of
Penicillium spp. peaked in spring (Fig. 3, Table 1). Similarly, Aspergillus
species spores were most numerous in summer and fall. The seasonal
variation pattern of Cladosporium, Penicillium, Alternaria, Fusarium,
Aspergillus and Trichoderma could be affected by the presence of avail­
able substrata. The observed increase in propagule concentration is
obvious for plant pathogens (such are Cladosporium, Alternaria, Fusarium
and Botrytis species) during the warmer seasons of the year, i.e. during
the vegetation period, indicating that the indoor air was probably
influenced by outdoor air influx (Fig. 3b). A similar seasonal distribution
for Cladosporium spores was reported by Sautour et al. (2009) in a
hospital environment, and by de Ana et al. (2006) for Alternaria species
in residential homes. It should be noted that research on the seasonal
spore variation of different fungal taxa is scarce. This amplifies the
importance of our work since it contributes to the knowledge of both the
diversity and seasonal distribution patterns of different fungal genera.
The xerophilic or xerotolerant fungi documented in this research
(notably Aspergillus penicillioides, members of the A. versicolor group, as
well as Eurotium species) play an important role in the deterioration of
cultural heritage artifacts (Michaelsen et al., 2006; Sterflinger and Piñar,
2013; Micheluz et al., 2015). Namely, in conditions of low water
availability, which are often present on cultural heritage artifacts, some
of these micromycetes could cause both esthetic and structural alter­
ations. Noteworthy are cases of foxing phenomena in paper materials
caused by the growth of A. penicillioides and Eurotium herbariorum (Arai,
2000). Furthermore, Arthrinium, Fusarium and Trichoderma species are
producers of cellulolytic enzymes (Sukumaran et al., 2005; Elsababty
et al., 2015) and therefore some of them are reported as degradation
agents of documentary heritage (Michaelsen et al., 2006). Additionally,
numerous species are known as producers of proteolytic enzymes,
including Aspergillus, Penicillium, Fusarium and Trichoderma species
(Michaelsen et al., 2006; Ljaljević Grbić et al., 2014; Unković et al.,
2018b). The importance of these species in the deterioration of pro­
teinaceous substrata of cultural heritage artifacts (such are parchment,
silk and wool fabrics or paint layer adhesives) must never be under­
estimated (Unković et al., 2018b; Savković et al., 2019). The production
6
International Biodeterioration & Biodegradation 157 (2021) 105163
of acidic metabolites is also demonstrated for many reported species,
and plays a significant role in stone monument deterioration. Namely,
excreted acids react with different ions in the substrate, leading to bio­
corrosion and secondary mycogenic mineral formation (Savković et al.,
2016). Furthermore, pigment production is reported for a vast number
of fungal species, which is the main cause of various esthetic changes in
artworks (Unković et al., 2018b; Savković et al., 2019). Many of the
isolates reported in our study are dematiaceous species that produce a
dark-colored melanin pigment, while extracellular pigment production
was additionally reported for others (Savković et al., 2019). Several
authors ascertained a strong correlation between aerobiological in­
vestigations and biodeterioration of cultural heritage objects, because
air is the main vehicle for the dispersion of microorganisms (Sorlini,
1993; Saiz-Himenez and Gonzales, 2007). Viable bacterial and fungal
loads in the air inside cultural heritage premises have been identified as
a major problem for the conservation of artworks (Lazaridis et al.,
2015). Furthermore, it is considered that airborne microorganism
composition is similar or identical to that isolated from cultural heritage
artifacts (Saiz-Himenez and Gonzales, 2007). Previously reported data
showed that although a direct impact on stored artifacts was not
observed, in numerous Aspergillus, Penicillium and Talaromyces isolates a
biodeterioration capacity was observed in vitro via applied biodeterio­
ration tests (Savković et al., 2019). According to these data, many tested
isolates have demonstrated significant deterioration capacity in vitro, i.e.
with the production of extracellular acids, enzymes and pigments. This
would allow us to ascertain a potential impact on cultural heritage ar­
tifacts. The measured indoor fungal load and microclimatic conditions
could represent a serious problem for works of art and are regarded as an
early indicator of fungal accumulation and biodegradation of cultural
heritage objects (Awad et., 2020). The presence of fungi, members of
different ecological groups, indicates that in aeromycological studies it
is necessary to use different isolation media to adequately assess both
species diversity and the concentration of viable fungal propagules. The
presence of xerophilic and cellulolytic fungi, due to their biodeteriora­
tion potential, should not be ignored in cultural heritage conservation
facilities.
It is considered that measuring the spore loads of certain species is
more important for the estimation of health risks than measuring total
spore concentrations (Garret et al., 1998). Therefore, identification of
airborne fungal isolates is crucial in assessing potential health hazards
for the people occupying certain facilities. Numerous studies infer that
respiratory allergies and asthma are correlated with the spore concen­
trations of specific fungal taxa, notably Penicillium and Alternaria species
(Licorish et al., 1985.; Garret et al., 1998; Bush et al., 2006). Further­
more, exposure to spores of different fungal species may result in severe
respiratory infections (respiratory mycoses), which are mainly reported
in immunocompromised patients (Khan and Karuppayil, 2012). Special
attention is given to certain Aspergillus species (notably A. fumigatus,
A. niger and A. flavus), which are regarded as important causative agents
of opportunistic respiratory infections in humans (Herbrecht et al.,
2004). Aspergilli, Penicillia and Fusaria are also well-known mycotoxin
producers (Bennet and Klich, 2003; Samson et al., 2004, 2010).
Although the presence of certain species is undoubtedly important,
measurements of total spore concentrations should not be disregarded
since guidelines concerning total fungal loads have been proposed by the
relevant specialized agency (World Health Organization, 1990; World
Health Organization Europe, 2009). However, it should be emphasized
that although a high amount of propagules was reported in the inves­
tigated premises, signs of severe health-related impacts are only
observed in employees exposed to fungal spores with average values
corresponding to 50,000 CFU m− 3 air (Hedenstierna et al., 1986;
Oppliger et al., 2005). To the best of the authors’ knowledge, respiratory
health issues were not reported for the staff in the cultural heritage
conservation facility in our study, which is in line with the prior state­
ment. Regarding various types of fungal contamination in cultural her­
itage conservation facilities and possible adverse health outcomes
Ž. Savković et al.
among employees, a systematic control of air quality is essential and
should become an integral part of hygiene-monitoring procedures
(Górny et al., 2016). It should be emphasized that the presence of certain
fungi in cultural heritage conservation facilities should never be
ignored.
5. Conclusions
Fungal diversity and propagule loads, within cultural heritage con­
servation premises, show significant and important seasonal variations.
As such, monitoring of presence and abundance of airborne fungal
deteriogens, can be a useful tool for an early indication in the risk
assesment of artworks biodeterioration.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was carried out as a part of projects Nos. 173032 and
176016 that were financially supported by the Ministry of Education,
Science and Technological Development of the Republic of Serbia.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ibiod.2020.105163.
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