International Biodeterioration & Biodegradation 54 (2004) 7 – 11

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Airborne and soil microfungi as contaminants of stone in a

hypogean cemetery
& & b
cov(aa;∗ , Miroslava G(odyov(aa , Jaroslav Sevc
Alexandra Simonovi&
a Faculty of Natural Sciences, Department of Soil Science, Comenius University, Mlynska dolina B2, Bratislava 842 15, Slovak Republic
b Faculty of Natural Sciences, Geological Institute, Comenius University, Mlynska dolina G, Bratislava 842 15, Slovak Republic
Received 10 May 2003; received in revised form 5 November 2003; accepted 20 November 2003

Abstract

In a hypogean Jewish cemetery in Bratislava containing cemetery soil and tombstones, the types of fungi on various calcite mineral
substrates in dark, cold and constantly moist chambers without direct ventilation and lighting were investigated. The mineral substrates
included monomineral limestone, and limestone containing quartz, magnesite, gypsum, micas, feldspar, illite and smectite. The character-
istics of the mineral substrates were examined by pH and X-ray-diphractographic analyses. The pH of the substrates in water ranged from
9.71 to 10.33. Structural changes in the substrates were revealed by scanning electron microscope photos. The samples yielded 36 di:erent
microfungi. The most common contaminants of the substrates analysed were Acremonium strictum, Alternaria alternata, Aspergillus
versicolor, Aureobasidium pullulans, Cladosporium sp., Fusarium sp., Penicillium sp., P. chrysogenum, P. viridicatum, and Trichoderma
sp. The investigation has shown that these hard, highly alkaline mineral substrates are susceptible to the growth of microscopic fungi,
sporulation and subsequent degradation of the materials.
? 2003 Elsevier Ltd. All rights reserved.

Keywords: Destruction; Calcite mineral substrates; Airborne and soil microfungi

1. Introduction Microscopic fungi, which exist in these extreme condi-

The biocorrosion of historical architecture and coloniza-
tion of many di:erent objects of natural or synthetic origin
is also within the sphere of microscopic fungal activities
&
(de la Torre et al., 1990; Kov(ac& ik, 2000; Simonovi& cov(a
et al., 2000; SterAinger, 2000). Although microscopic
fungi are small, their surface area is signiBcantly larger
than, for example, bacteria, and their hyphae are able
to attack and penetrate mineral substrates (Fig. 1). Var-
ious workers (Diakumaku et al., 1995; Saiz-Jimenez,
1995; Saiz-Jimenez et al., 1995) consider that the metabo-
lites of microscopic fungi (melanins, melanoids and intra-
cellular polymerizing products) cause pigmentation of mar-
ble, sandstone, limestone and other materials. The chemical
inAuence of these compounds creates defects in the struc-
ture of the materials, e.g. micro- and macroscopic cracks,
deformations, roughness, and black stains and crusts. The
Bnal result of these processes is reduction of porosity and
the loss of their original character.
∗ Corresponding author.
tions, have not been well characterized yet. It is therefore
very important to determine those fungal genera growing
on such mineral substrates and study their biology in order
to understand the mechanisms of the adaptation processes.
This is necessary for e:ective protection against biodeteri-
oration of mineral materials by microscopic fungi. In the
present study, the types of mould present on various calcite
mineral substrates in dark, cold and constantly moist cham-
bers of a hypogean Jewish cemetery in Bratislava without
ventilation and lighting were investigated together with ev-
idence of biocorrosive and destructive activity.
2. Material and methods
Samples of stone were collected for mineralogical analy-
ses by scalpel from markedly damaged surfaces with visible
colonies of microscopic fungi and sliced until 0:12 mm thick
(c. 0:1 g) before preparation for X-ray-diphractographic and
pH analyses. The samples were subjected to mechanical
agitation for 2 min in distilled water (ratio of solid:water,

0964-8305/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2003.11.004
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A. Simonovi/
cova et al. / International Biodeterioration & Biodegradation 54 (2004) 7 – 11
Fig. 1. Hyphae of fungal mycelium penetrating into the sub-surface
structure of a sample of deteriorated stone.
1:10) in an electromagnetic mixer operating at 120 rpm.
After the samples were dried at room temperature and coated
with gold–palladium, micrographs of the damaged substra-
tum were taken on a JEOL (JXA-840) scanning electron
microscope (SEM).
The samples for mycological analyses were collected
from the same stone objects as for mineralogical analyses by
swabbing with sterile cotton swabs. In the laboratory, swab
samples were shaken mechanically for 10 min in 10 ml
sterile distilled water and 1 ml aliquots of the resulting
suspensions used to prepare spread plates on Czapek-Dox,
potato-dextrose, malt extract and chloramphenicol yeast
glucose agar (HIMEDIA, Bombay) in order to isolate as
wide a range of microfungi as possible. Plates were in-
cubated in the dark at laboratory temperature (25◦ C) for
14 days and the microscopic fungi were identiBed using the
diagnostic keys of Pitt (1991), de Hoog and Guarro (1995),
Samson et al. (1996) and Domsch et al. (1980).
3. Results and discussion
The results of mineralogical analyses are shown in
Table 1. Samples 2, 3, 6 and 10 are almost monomineral
Table 1
Mineralogical composition and pH of various stone substrates
Samples Major components
2 Calcite (CaCO3 )
3 Calcite
6 Calcite
10 Calcite
7 Calcite
9 Calcite
5 Calcite, quartz
8 Calcite, quartz
1 Quartz, calcite
4 Quartz, calcite
a Measured 1h after addition of stone sample to distilled water.
Fig. 2. Mycelium and conidia of Aspergillus versicolor in the coating on
the surface of a sample of deteriorated stone.
(calcite), containing only small amounts of quartz. Samples
7 and 9 contained principally calcites, with quartz as a sec-
ondary component and minimal amounts of magnesite and
gypsum. In samples 5 and 8, the amount of quartz was sub-
stantially greater and smaller amounts of micas, feldspars
and illite were also present. Quartz and calcite dominate in
samples 1 and 4 and micas, feldspar and illite are present in
smaller amounts.
Although the minerals were highly alkaline, the pH
of the water in the steeped samples being 9.71–10.33
(Table 1), viable micromycetes were present and evidently
able to grow on the minerals, creating mycelial networks
(Fig. 1) and also producing conidia (Fig. 2). The presence
of microscopic fungi on such alkaline substrates is indica-
tive of their ability to modify the pH of the environment
by producing organic acids and other acidic metabolites.
Mycological analysis conBrmed that visible coatings on the
surface of the various types of stone were of fungal origin.
Structural changes in mineral substrates caused by micro-
fungi were revealed by SEM (Figs. 1 and 2). Fig. 1 shows a
mineral substrate which has been subjected to the extended
inAuence of adverse microclimatic conditions (elevated hu-
midity) that support biodeterioration of the stone surface by
Minor components pHa
Quartz (SiO2 ) 9.94
Quartz 9.84
Quartz 9.83
Quartz 10.33
Quartz, gypsum(CaSO4: 2H2 O) 9.71
Quartz, magnesite (MgCO3 ) 10.18
Various types of mica, feldspar, illite 9.94
More than one type of mica, feldspar, illite, smectite 9.95
More than one type of mica, feldspar, illite 10.27
more than one type of mica, feldspar, illite 9.76
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A. Simonovi/
cova et al. / International Biodeterioration & Biodegradation 54 (2004) 7 – 11 9

microscopic fungi, with the fungi spreading over the sur-

Fig. 3. Undamaged surface of a sample of stone showing idiomorphic
crystals of quartz with calcite (Collection of Faculty of Natural Sciences,
Comenius University, Bratislava).
face and their hyphae penetrating into the structure. As can
be seen in Fig. 2, they are also able to sporulate in an envi-
ronment deBcient in organic matter as they slowly destroy
the mineral substance (Fig. 2). In comparison, Fig. 3 shows
a substrate (from the Collection of Faculty of Natural Sci-
ences in Bratislava) with aggregates of idiomorphic crystals
of calcite and quartz undamaged by the activities of micro-
fungi.
Altogether, 36 species of microfungi were isolated on
the four di:erent agar media employed, illustrating the
gross contamination of the damp, dark chambers where the
stone samples were collected. The range of types isolated
is typical of a soil mycobiota (Table 2), but it is distinctly
possible that the minerals may have been contaminated by
airborne spores rather than by direct contamination from
soil in the moist, dark chambers where the stone sam-
ples were collected. With increasing quartz content in the

Table 2
Microfungi isolated (*) from deteriorated stone samples

Micromycetes Mineral substrates

2 10 6 3 9 7 8 5 4 1
Acremonium sp. * * * * *
A. butyri * *
A. strictum * * *
Alternaria sp. * *
A. alternata * * * * *
A. tenuissima * * *
Arthrobotrys sp. *
Aspergillus fumigatus *
A. versicolor * *
Aureobasidium pullulans * *
Cladosporium sp. * * *
C. cladosporioides *
C. herbarum *
Clonostachys rosea *
Doratomyces sp. *
Fusarium sp. * * * * *
F. solani *
F. sporotrichioides *
Gliocladium sp. *
Mucor sp. * *
M. hiemalis f. hiemalis * *
Paecilomyces lilacinus *
Papularia sp. *
Penicillium sp. * * * * * * * * *
P. aurantiogriseum *
P. brevicompactum * *
P. chrysogenum * * * *
P. expansum *
P. griseofulvum * *
P. oxalicum *
P. viridicatum * * * * * *
Scopulariopsis sp. *
Trichocladium asperum *
Trichoderma sp. * * * * *
T. viride * *
Trichophyton sp. * *
Total: 36 species 13 2 9 1 15 9 10 9 7 9
19 species 20 species 17 species 14 species
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A. Simonovi/
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Table 3
Dominant species of airborne and soil microfungi on the mineral substrates and their acid production and staining (after Domsch et al. (1980), Gravesen
et al. (1994))
Soil microscopic fungi Acid production
Alternaria alternata Various fatty acids and fatty acid esters
A. tenuissima Glutamic acid
Aspergillus versicolor Norsolorinic acid, many toxic metabolites
Aureobasidium pullulans Fumaric, ferulic, gluconic, oxalic, oxaloacetic acid
Cladosporium sp.
Fusarium sp. Acetic and citric acid
Penicillium sp. Citric and oxalic acid
P. chrysogenum Citric, gluconic, and -ketoglataric acid
P. viridicatum Viridic acid
Scopulariopsis sp. SpeciBc metabolites
Trichoderma viride Various biologically active or toxic compounds
mineral substrates, the range of micromycetes isolated
decreased (Table 2), but no other connection between the
composition of the mineral substrates and micromycete bio-
diversity was observed. These results indicate a preliminary
relationship, however, and the inAuence of the substrate
character on the composition of the associated micromycete
community is still an open question.
Acremonium sp., Alternaria alternata, Penicillium sp.,
Penicillium viridicatum and Trichoderma sp. were com-
mon contaminants of all the substrata analysed. Mycologi-
cal analysis conBrmed that the most abundant and dominant
species of soil microfungi were: Acremonium strictum,
Aspergillus versicolor, Aureobasidium pullulans, Cla-
dosporium sp., C. cladosporioides, Fusarium sp., Peni-
cillium sp., Penicillium chrysogenum, P. viridicatum, and
Trichoderma sp. Species of Alternaria were also among
the dominant microfungi on the mineral substrates, six of
the ten minerals being contaminated by members of this
genus. Diakumaku et al. (1995) considered the species of
Alternaria as being the main cause of brown and black
stains on marble, limestone and sandstone of many di:erent
monuments in Europe and Africa. Although the genus con-
tains plant parasites, a few species are ubiquitous and very
frequently soil-borne. A. alternata is the commonest of
these. This species is well adapted to cold conditions, with
the minimum temperature for growth ranging from −5◦ C
to 0◦ C. Maximum growth occurs at pH 4 –5.4, but growth
is possible over the pH 2.7–8.0, according to Domsch et al.
(1980). A. alternata is therefore acidophilic, whereas the
pH of the mineral substrates from which it was isolated var-
ied, but was highly alkaline. The presence of A. alternata
and A. tenuissima on and in such highly alkaline mineral
substrates is therefore very interesting. A. alternata has also
been isolated from coatings on aragonite in some caves at
Zbra&sov, Czech Republic, where it dominated together with
P. aurantiogriseum, P. chrysogenum, A. versicolor and
Cladosporium sp. (Marvanov(a et al., 1992). A. alternata
Staining
Dark brown
Dark brown
Green
Yellow, red, purple, environmental isolates often
olivaceous-green, black
Olivaceous-brown, blackish brown
Red, vinaceous
Green, greenish-blue, dull green
Yellow, green
Green
Brown o black
Green
is also one of the most important allergenic fungi causing
asthma (Gravesen et al., 1994). The species of Acremo-
nium, which were very frequent in the mycobiota of the
mineral substrates examined in the present study (A. butyri,
A. murorum and A. strictum), are typical soil microfungi.
The high frequency of their presence in mineral substrates
is paradoxical, since they grow most rapidly under acid
environmental conditions like Alternaria.
It is also interesting that among the microfungi isolated
from the mineral substrates are species that have been pre-
viously reported as causing pigmentation of substrata and
producing organic acids, which could cause corrosion. As
shown in Table 3, most of the commonest species isolated
from the mineral substrates (Aspergillus versicolor, Aure-
obasidium pullulans, Alternaria alternata, P. chrysogenum,
etc.) are producers of many di:erent acid metabolites and
exogenous pigments causing staining.
Acknowledgements
We would like to thank Dr. J. Stankovi&c and his col-
leagues for their assistance in the SEM investigation (Central
Laboratory of Electron Microscopy of the Faculty of Sci-
ence, Comenius University, Bratislava). This research was
supported by ScientiBc Grant Agency VEGA 9114/02 and
9118/02.
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