SPECIAL ISSUE
Ascomycete fungi on dimension stone of the ‘‘Burg Gleichen’’,
Thuringia
Christine Hallmann • Diana Fritzlar •
Lorena Stannek • Michael Hoppert
Received: 7 January 2011 / Accepted: 22 April 2011 / Published online: 13 May 2011
 The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract In the present study, the diversity of ascomycete
fungi was investigated on two wall areas of the ‘‘Burg
Gleichen’’, Thuringia (Germany), made of various types
of sandstones, travertine and Grenzdolomit. From a
W-exposed, shaded wall area, free-living ascomycetes
(mainly ‘‘black fungi’’) and green algae could be retrieved
from sandstone lithologies. Sandstone from an ESE-exposed
area was mainly colonized by lichen ascomycetes and the
lichen alga Trebouxia. Both areas share a small number of
generalist species, related to the ascomycete black fungi
Sarcinomyces petricola, Phaeococcomyces chersonesos and
Stichococcus mirabilis. Free-living black fungi were isolated
and characterized with respect to cell wall morphology
and melanin content. A remarkably rigid melanin layer,
incorporated in the cell wall of a Cladosporium isolate is
presented in detail.
Keywords Dimension stone  Biofilm  Ascomycete
fungi  Green algae  Cell wall  Melanin
Introduction
Ascomycete fungal organisms are most successful colo-
nizers of all terrestrial habitats, such as rock and soil (e.g.
Gorbushina and Krumbein 2000; Anderson and Cairney
2004). They interact in different ways with other organ-
isms, as symbionts (in mycorrhiza or lichen symbiosis), as
pathogens or as important destruents of most organic
compounds, especially of plant litter (Hattenschwiler et al.
2005). Although they are heterotrophic, i.e. they depend on
organic substrates produced by other organisms, many of
them are adapted to low availability of nutrients (Wainright
et al. 1993). Moreover, fungal spores are resistant to des-
iccation and radiation. Owing to their small size, the spores
are dispersed by a variety of vectors, in particular by wind,
but also by animals and by plant diaspores.
Fungal hyphae penetrate surfaces and grow inside soil, but
also in various types of clastic rocks (e.g. sandstone) and
homogeneous material, e.g. dolomitic limestone (Gorbushina
et al. 1993, Hoppert et al. 2004, Gorbushina 2007). The
fungal hypha is among the fastest growing cell in nature:
hyphae grow with a velocity between 10 and 40 lm/min
(Trinci and Saunders 1977). An active growth zone of
10–15 lm extends behind the apex (Steinberg 2007). The
active growth zone is also important for surface adhesion, the
excretion of lytic enzymes (e.g. for penetration of wood) and
the exertion of mechanical forces on the surface. For endo-
lithic growth, active dissolution of calcitic matrices by
organic acids (e.g. Ascaso et al. 1998) is one important
mechanism for active penetration. Fungal hyphae find a path
in sedimentary material (cf. Fig. 1) by growing around small
particles. This feature is brought about by fast changes in the
growth direction of the hyphal tip and could also be observed
in organisms of other phyla, such as plant root hairs or
streptomycetes (Geitman and Emons 2000; Fla¨rdh 2003).
C. Hallmann
Abt. Experimentelle Phykologie und Sammlung fu¨r
Algenkulturen, Albrecht-von-Haller-Institut fu¨r
Pflanzenwissenschaften, Georg-August-Universita¨t Go¨ttingen,
Untere Karspu¨le 2, 37073 Go¨ttingen, Germany
D. Fritzlar  L. Stannek  M. Hoppert (&)
Institut fu¨r Mikrobiologie und Genetik,
Georg-August-Universita¨t Go¨ttingen,
Grisebachstraße 8, 37077 Go¨ttingen, Germany
e-mail: mhopper@gwdg.de
M. Hoppert
Courant Research Centre Geobiology,
Georg-August-Universita¨t Go¨ttingen,
37077 Go¨ttingen, Germany
123
Environ Earth Sci (2011) 63:1713–1722
DOI 10.1007/s12665-011-1076-y
Ascomycete fungi on building stone are well known and
have been frequently described, mostly in context to bio-
deterioration (e.g. Gorbushina et al. 1993; Diakumaku et al.
1995, Sterflinger et al. 1997; Gorbushina and Krumbein
2000; Simonovicova et al. 2004). One important group of
colonizers, the Dematiaceae, produce melanin in vegetative
hyphae (Cooke 1961; Wollenzien et al. 1995). Though all
fungi produce various types of pigments, melanin is pre-
dominant in cell walls of reproductive structures, such as
conidia or fruiting bodies, including all types of spores
(Bell and Wheeler 1986). The melanisation of vegetative cells
in Dematiaceae leads to a heavy black pigmentation of all
colonized surfaces (Saiz-Jimenez 1995). The frequently
described meristematic growth form of Dematiaceae with
spherical instead of filamentous cells may be an additional
adaptation to adverse growth conditions in rock and stone
habitats, e.g. desiccation stress (Wollenzien et al. 1995).
The presented study is part of a comparative analysis of
microbial communities from two natural stone walls from the
Burg Gleichen (cf. Stu¨ck et al. 2011 [this issue]). Certain
differences in the abundance of cyanobacteria on wall sections
have already been described (Hoppert et al. 2010). Here, we
show the occurrence of ascomycete fungi on two wall sec-
tions. Typically, the fungal isolates produce melanin in vege-
tative cells. These melanin deposits form distinct layers,
extractable from cell walls of one Cladosporium isolate.
Materials and methods
Sampling
The sampling sites were several wall sections of the Burg
Gleichen, near Gotha, Thuringia (505204900 N, 105002000 E).
Samples (approx. 100 ll dry volume per sample) were collected
in May 2009 from ESE (wall area A) and W-exposed (wall area
B) walls with different lithologies (Fig. 2). The samples were
scraped off with a sterile scalpel and collected in sterile 2-ml
reaction tubes. Samples for clone libraries were randomly taken
from sites, where sufficient material could be removed with a
scalpel. All samples in area A were taken from the wall base
(Fig. 2) made of either Gleichenberger Ra¨tsandstein or Sem-
ionotussandstein. Wall joints were mostly closed, i.e. filled with
mortar. Area B exhibited a variety of lithologies (travertine,
Grenzdolomit, Ra¨tsandstein). Most wall joints were open.
Samples from Ra¨tsandstein from the wall base were taken into
consideration for this study. Each sample was labeled with either
A or B (according to the wall section) and with a respective
running code number. The subset of samples from sandstone
lithologies was processed for the detection of fungal clones.
Cultivation and isolation
For the enrichment and isolation of filamentous fungi, small
amounts of the biofilm samples were used to inoculate culture
media prepared according to Staley 1968, with modifications
(peptone/yeast extract/glucose medium, PYG): Bacto-peptone
0.25 g/l, yeast extract 0.25 g/l, glucose 0.25 g/l, basal salt
solution 19 ml/l, trace element solution 1 ml/l (basal salt
solution in 500 ml: nitrilotriacetic acid 3.0 g, MgSO4 9
7H2O 7.2 g, CaCl2 9 2H2O 1.7 g (NH4)6Mo7O24 9 4H2O
3.0 mg, FeSO4 9 7H2O 49.5 mg. Trace element solution in
100 ml: EDTA 250.0 mg, ZnSO4 9 7H2O 1.1 mg, MnSO4 9
1H2O 154.0 mg, FeSO4 9 7H2O 500.0 mg, CuSO4 9 5H2O
39.2 mg, CoSO3 9 7H2O 19.6 mg, Na2B4O7 9.34 mg). For
solid media, 1.5% (w/v) Bacto-Agar was added. Isolation of
representative strains was performed by repeated streaking either
single colonies or conidia on agar plates until apparent macro-
scopic and microscopic homogeneity of colonies and conidia.
Isolates were incubated about 2 weeks at room temperature
under ambient light. Liquid cultures were incubated in a shaking
water bath in the dark. For some growth experiments, a piece of
Seeberger Sandstein was grounded in a mortar and sterilized by
autoclaving. Equal volumes of isolated strains were mixed with
PYG agar medium (kept liquid at 95C). The mixture was
applied on a sterile microscopic slide and inoculated after
solidification. The samples were inspected by light microscopy
(Axioscope 40, including Axiocam MRm, Carl Zeiss Micro-
imaging, Go¨ttingen).
DNA extraction, PCR, cloning and sequencing
For isolation of environmental DNA as well as DNA from
fungal isolates, cloning and sequencing steps were per-
formed as already described (Hallmann et al. 2010 [this
issue]) with following modifications: For two samples
(A1-4 and B18) cell disruption for 30 s and 50 s of beat
Fig. 1 Growth experiment with a Cladosporium isolate. a Growth in
agar medium without solid particles. b Growth in agar medium mixed
with of sandstone clasts. Hyphae are traced (white lines). Note the
rapid changes of growth directions as compared with a
1714 Environ Earth Sci (2011) 63:1713–1722
123
beating was applied (if not especially mentioned, results of 30 s
beat beating were shown). For identification of ascomycetes,
the primer pair ITS1 (50-TCCGTAGGTGAACCTGCGG) and
ITS4 (50-TCCTCCGCTTATTGATATGC) for the ITS (inter-
nal transcribed spacer) were used (Anderson and Cairney
2004). Accession numbers of sequences related to this study are
deposited online (GenBankTM: http://www.ncbi.nlm.nih.gov/
genbank) with this publication as a reference.
Electron microscopy
For electron microscopy, vegetative filaments were either
taken from solid or liquid cultures, concentrated by filtering
over a 0.45-lm size pore filter and embedded in resin according
to Spurr (1969). Agar blocks were chemically fixed overnight
in 2.5% (v/v) glutaraldehyde (grade I for electron microscopy,
Sigma–Aldrich, St. Louis, MO, USA) and for 2 h in osmium
tetroxide (1%, w/v, aqueous solution, Science Services,
Munich). Samples were dehydrated in a graded ethanol series
and infiltrated with Spurr resin over 48 h before polymerization
at 70C for 12 h. Ultrathin sections of 80-nm thickness were cut
with glass knives. Post-staining of sections was performed with
1.5% (w/v) phosphotungstic acid for 5 min (Hoppert 2003).
Electron microscopy was performed with a Zeiss EM 902
transmission electron microscope, equipped with a 1 K digital
camera (Carl Zeiss NTS, Oberkochen).
Fig. 2 Wall areas of the Burg
Gleichen selected for sampling.
a Wall area A (Romanesque
Hall). b Wall area B (section of
the ring wall). Sampling sites
are marked by red squares. The
schematic drawing (original
figure: Wanja Wedekind) shows
the location of the walls in the
castle complex
Environ Earth Sci (2011) 63:1713–1722 1715
123
For immunogold labeling with the lectin concanavalin A
(Con A), the protocol according to Hallmann et al. 2010
(this issue) was applied. Gold markers in images were
digitally enhanced as described (Hoppert and Holzenburg
1998).
Melanin preparation for electron microscopy was per-
formed according to Rosas et al. 2000, with modifications.
A melanised Cladosporium isolate was cultured on PYG-
medium and then centrifuged for 30 min at 3,0009g. The
cells were washed in 100 mM potassium-phosphate buffer,
supplemented with 0.9% (w/v) sodium chloride (phos-
phate-buffered saline, PBS). Then, cells were resuspended
in 1.0 M sorbitol/0.1 M sodium citrate solution (pH 5.5),
containing 10 mg/ml lysis enzyme from Trichoderma
harzianum (Sigma–Aldrich) and incubated at room tem-
perature overnight. Cells were again centrifuged, resus-
pended in PBS and incubated in 4 M guanidinium
thiocyanate solution for 12 h at room temperature. After
washing the cells in PBS by centrifugation and resuspen-
sion in reaction buffer (10 mM Tris, 1 mM CaCl2, 0.5%,
w/v SDS), 1 mg/ml final concentration proteinase K was
added. The suspension was incubated for 4 h at 65C. Cells
were again washed in PBS and boiled for 6 h in 6 M
aqueous HCl solution. The remaining melanin preparation
was dialyzed for 2 days against distilled water (Visking
dialysis tubing, molecular weight cutoff 12,000–14,000,
Serva, Heidelberg). The prepared melanin was then sub-
jected to embedding in Spurr resin as described above.
Results
Both areas sampled (Fig. 2) exhibited obvious colonization
by green algae (in addition, cyanobacteria on area A) and
epilithic or endolithic lichens (cf. Fig. 3). Approximately,
one-third of the collected samples was useful for cloning of
fungal genera. For generation of fungal clone libraries,
sequencing with ITS primers turned out to be most
appropriate (Anderson and Cairney 2004). From areas A
and B, 65 (48) and 30 (75) fungal (algal) clones were
retrieved, respectively. It has to be kept in mind that the
organisms were identified by their sequences of closest
known relative by BLASTn analysis (National Center for
Biotechnology Information, Bethesda, MD, USA). The
genus and species names given in Tables 1 and 2 represent
the closest known relative according to these sequence
similarities.
Table 1 lists the fungi identified from the clone libraries,
along with the algal clones. Mostly, the apparent coloni-
zation correlates with clone bank data. All sites from area
A were apparently colonized by endolithic lichens.
Accordingly, a major part of algae and fungi retrieved from
the clone banks were, in fact, lichen associated. Samples
B13, B14 and B18 were taken from sites apparently colo-
nized by green algae. The (non-lichen associated) algae
Stichococcus and Pseudochlorella could also be retrieved
from the clone banks. In addition, numerous Sarcinomyces
and Phaeococcomyces-related fungal clones were present.
Because the cell disruption method may have detri-
mental effects on the quality of clone libraries (i.e. with
respect to species richness), different intensities of bead
beating were applied on two samples. The data in Table 1
(sample B18 30 s and 50 s beat beating time; sample A1-4
30 s and 50 s) document a minor variation in the retrieved
clones, but do not show the occurrence of essentially dif-
ferent species groups. Thus, both pairs of samples could be
assigned to either wall area A or wall area B-typical groups
of clones.
Although the retrieved clone banks also differ from
sample to sample, two groups of species typical for either
of the wall areas can be clearly distinguished. It is obvious
that the fungal diversity on area A is higher when com-
pared with area B, where more algal than fungal species
could be identified. Just a small number of identical species
(represented by the respective clones) were present in both
Fig. 3 Macroscopic appearance of (microbially) colonized sandstone
surfaces. a Endolithic lichens in conjunction with scaling and
backweathering of the surface. b Layer of green algae
1716 Environ Earth Sci (2011) 63:1713–1722
123
areas: representatives of two ascomycete fungi, Sarcino-
myces petricola- and Phaeococcomyces chersonesos- as
well as Stichococcus mirabilis-clones (green algae) were
retrieved with high clone numbers. One lichen ascomycete
(from Caloplaca decipiens) and a Trebouxia sp. lichen alga
were found in samples from both walls, but just in low
numbers. In particular, the fungal clones in both groups of
samples are different (Fig. 4). On area A, most ascomy-
cetes are related to genera known from lichen symbiosis
(cf. Wirth 1995). Also the presence of several lichen alga
clones (Trebouxia) reflects this feature. This is also in
accordance with the observation that mainly at the wall
base, the sandstone was intensively colonized by endolithic
lichens.
Although also area B exhibited colonization by endo-
lithic lichens at the wall base, mainly non-lichenized
organisms could be retrieved: Here, just one clone of the
typical lichen alga and two lichen fungi clones could be
found. Among the fungal organisms, especially Sarcino-
myces and Phaeococcomyces clones were retrieved,
Table 1 Distribution of fungal and algal clones on wall sections
Closest relative species A2-1 A1-4 
A1-4 
(50 s) A1-8 B13 B14 B17 B18 
B18 
(50 s) B22
% 
sequence 
similarity 
to closest 
relative 
species
Sarcinomyces petricola * 1 1 1 6 5 2 5 97-98
Phaeococcomyces chersonesos * 2 2 3 1 4 1 1 96-97
Uncultured Cladosporium clone * 1 1 99-100
Caloplaca decipiens 1 2 99
Capnobotryella sp. * 6 3 99
Cladosporium cladosporioides* 1 1 99
Phaeobotrysphaeria citrigena 1 90
Uncultured Dothideomycetes 3 10 82-83
Anisomeridium polypori 16 1 86-88
Dolichousnea longiss ima 1 96
Phaeophyscia ciliata 8 89-90
Pseudocyphellaria fimbriatoides 1 83
Umbilicaria arctica 1 85
Erysiphe alphitoides 1 99
Pleospora herbarum 1 100
Stichococcus mirabilis 1 7 4 16 7 5 15 8 7 89-96
Trebouxia sp. 1 1 98-99
Uncultured Trebouxia photobiont 4 1 11 93-100
Trebouxia arboricola 1 97
Chlorella sp. 1 92
Stichococcus related 1 2 18 5 1 79-82
Pseudochlorella sp. 1 1 2 2 86-89
Black fungi are marked by an (*), lichen algae and lichen fungi are marked in red
Boxed areas indicate species present in both wall areas (green), exclusively present in area A (blue) or in area B (beige)
The predominance of Sarcinomyces petricola in area A is indicated by a dark green color
Table 2 BLAST search results
of fungal isolates
Sample no. Closest relative species, accession no. Percentage sequence similarity
to closest relative species
B 1-2 Beauveria bassiana, GQ302680 100
A 1-2, B1-3, B12, B 2-5, B10 Cladosporium cladosporioides strain F12,
HQ380766
99–100
B 1-8, B14 Phaeococcomyces chersonesos, AJ507323 96
Environ Earth Sci (2011) 63:1713–1722 1717
123
i.e. representatives of free-living, non-lichenized black
fungi. The overall diversity was considerably lower (10
different blast responses) than on area A (19 different blast
responses).
Several ascomycetous black fungi could be isolated
from the samples, especially from area B, as listed in
Table 2. Although the diversity of ascomycete clones was
higher on area A, just one fungal isolate could be obtained
from this area. Since the abundant lichen ascomycetes from
area A are rather difficult to culture in standard media, they
could not be retrieved as pure cultures. Several Clado-
sporium and Phaeococcomyces-related isolates could be
obtained from area B. Though just one Cladosporium-
related clone could be retrieved from clone banks, several
Cladosporium strains could be isolated, accounting for a
high abundance of diaspores, but a low number of actively
growing organisms at the time of sampling.
All isolates belong to the black (melanised) fungi and
served as model organisms for further studies like obser-
vation of melanin production and growth experiments. The
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
wall area A wall area B
ascomycetes
lichen fungi
lichen algae
green algae
Fig. 4 Relative amounts of clone numbers of large phylogenetic
groups in wall areas A and B
Fig. 5 Melanin production in Cladosporium and Phaeococcomyces
strains. Cladosporium (a) and Phaeococcomyces (b) on solid
medium. c Cladosporium strain in liquid culture; vegetative hyphae
form globules due to culturing conditions and show a dark stain.
d Growth of Cladosporium without formation of melanin after several
culture passages
1718 Environ Earth Sci (2011) 63:1713–1722
123
growth experiment in Fig. 1 illustrates that Cladosporium,
like all other isolates, is able to grow invasive in agar
media containing a suspended fraction of crushed sand-
stone. One ascomycete (Beauveria bassiana) could be
obtained in pure culture, but not from the clone banks. The
formation of melanin in solid and liquid cultures is shown
in Fig. 5a–c. The melanised isolates exhibit thick, multi-
layered cell walls (Fig. 6). In Phaeococcomyces, two
chemically distinct cell wall layers could be observed: the
inner layer is labeled by the lectin marker concanavalin A
(cf. Hallmann et al. 2010 [this issue]), i.e. the Con A-gold
particles bind to this feature. An outermost layer consists of
less densely packed extracellular polymers (exopolymers;
Fig. 6a, b). In Cladosporium isolates, thick layers of
exopolymers between adjacent cells could be observed
(Fig. 6c). The walls are approximately 400 nm in thick-
ness (Fig. 6d). A liquid culture shown in Fig. 5d illustrates
that melanin production may also get lost after several
passages of culturing in the laboratory. There is a striking
difference in cell wall thickness between melanised and
non-melanised Cladosporium isolates (Fig. 7). The non-
melanized cell wall appears by a factor of 10 thinner,
without any sign of exopolymer formation (Fig. 7b).
Although the melanised cell wall appears thick, the
melanin itself represents just a small, but very rigid pro-
portion of the cell. While it was not possible to extract
melanin from the Cladosporium isolates, distinct melanin
cell wall fragments (‘‘melanin ghosts’’) from Phaeococc-
omyces could be retrieved. Here, the melanin represents
one distinct layer of the cell wall that could be isolated
after harsh treatment with proteolytic and glycolytic
enzymes as well as boiling in hydrochloric acid. Even after
this treatment, the original shape of the cell wall was still
preserved (Fig. 8).
Fig. 6 Phaeococcomyces and Cladosporium cell walls; transmission
electron microscopy of ultrathin sections. a, b Phaeococcomyces
showing a multilayered cell wall (arrows) with an outermost
exopolymer layer. b Detail with a Con A-gold labeled wall (arrows;
gold label encircled) and an unlabeled outermost exopolymer layer
(asterisk). Cladosporium strain: cross sections of three cells (c),
embedded in an extracellular matrix (asterisks). Multilayered cell
wall of a single cell (d)
Environ Earth Sci (2011) 63:1713–1722 1719
123
Discussion
This study is focused on ascomycete fungi, but the clone
libraries revealed also green algal clones. The selected
primer pair exhibits a broad specificity for ITS sequences
of fungal (as well as algal) organisms and has already been
successfully applied for a similar study (Berdoulay and
Salvado 2009). However, a phylogenetic analysis com-
prising all phylotypes within fungi (and other eukaryotic
microorganisms) would require a whole set of primers
(Anderson and Cairney 2004 and references therein). Thus,
though we could state clear differences between the wall
areas, the whole diversity of present phylotypes, as e.g.
analyzed in a study of rock-inhabiting fungi related to
Dothideomycetes, could not be retrieved (Ruibal et al.
2009). It should be noted that also the lack of sequence
information from well-characterized isolates in the public
databases limits the ‘‘taxonomic resolution’’ in our
approach (cf. Anderson and Cairney 2004). Hence, some-
times just low sequence similarities of the clones with
already known relative species could be retrieved (cf.
Table 1). Especially, the data on lichen ascomycete genera
must be interpreted carefully and shall be taken as an
indicator for the presence of ascomycetes involved in
lichen symbiosis, but not necessarily for the occurrence of
a defined lichen species.
Generally, the significant differences between ascomy-
cete fungi on different wall sections of the ‘‘Burg Glei-
chen’’ are obvious. Though all samples in this study were
taken from sandstones, some impact from surrounding
limestone lithologies (in area B), fine soil in wall joints and
mortars may influence directly or indirectly the situation
(pH, available nutrients or ions) for colonizing microor-
ganisms. Particularly, gypsum-containing mortar in wall
area A (cf. Hoppert et al. 2010) may have influenced
species diversity on this site. Moreover, open wall joints in
area B provide numerous small niches for depositions of
soil, moisture, bird droppings and other nutrient sources
that may influence microbial growth. These effects cannot
be completely excluded in most ‘‘field’’ situations, and are
difficult to quantify. There is, however, no direct evidence
for the influence of the limestone in area B. Otherwise,
more lichen fungal clones deriving from calcicolous and or
nitrophilous lichens should be expected (cf. Arino et al.
1997), which is, in fact, not the case. Generally, wall area
A is directly exposed to sunlight, and is therefore more
subjected to desiccation stress than the W-exposed wall
area B, directly located in the shadow of an adjacent tower
(cf. Fig. 2).
Clearly, lichen fungi and lichen algae represent a major
part of the microbial flora on the sun (ESE)-exposed wall
surface. At the W-exposed wall surface (area B), the non-
lichenized black fungi Sarcinomyces petricola and Phae-
ococcomyces chersonesos as well as Stichococcus mirabilis
and other Stichococcus-related algal clones could be
retrieved in high abundance, but clones of non-lichenized
ascomycetes as well as lichen-associated genera were rare.
Sarcinomyces and Phaeococcomyces were also present in
area A accounting for broad ecological amplitudes of these
organisms (cf. Wollenzien et al. 1997; Bogomolova and
Minter 2003; Michailyuk 2008; Hallmann et al. 2010 [this
issue]). Sarcinomyces, however, could only be retrieved in
low abundance from area A. It has to be discriminated
between the Stichococcus mirabilis-related clones (abun-
dant in both areas), and clones, more distantly related to the
genus Stichococcus (‘‘Stichococcus-related’’ in Table 1).
The latter group could be clearly assigned exclusively to
wall area B (cf. Table 1).
Fig. 7 Cell walls with and without melanin in comparison; trans-
mission electron microscopy of ultrathin sections. Cladosporium
strain, liquid culture. a Melanin-producing cell in cross section with a
multilayered cell wall (arrows) of several 100 nm in thickness (cf.
Fig. 5c). b Thin cell wall (between both arrows) of some tens of nm
(non-melanised variant, cf. Fig. 5d)
1720 Environ Earth Sci (2011) 63:1713–1722
123
These data imply that lichenized organisms are suc-
cessful competitors on area A, compared with some non-
lichenized green algae, but also compared with the black
fungus Sarcinomyces. The equal distribution of Phaeo-
coccomyces chersonesos- and Stichococcus mirabilis-rela-
ted clones in both areas indicate that some species remain
obviously completely unaffected. In wall area B, although
lichen colonization was observable, non-lichenized
fungi and algae dominated. It may be possible that the
rather moist and nutrient-rich conditions are more favor-
able for non-lichenized generalists (cf. Hoppert and Ko¨nig
2006).
Most of the ascomycetes, either identified by their
sequences or isolated from the stone surface are known as
melanised strains. Although melanisation is also known
from lichen ascomycetes, the non-lichenized genera have
been intensively studied with respect to their pigmentation
(e.g. Diakumaku et al. 1995). Besides endolithic growth (in
particular, the formation of micropits), pigmentation is an
obvious hazard on stone surfaces caused by these organ-
isms on stone surfaces. This effect is rather important for
the appearance of smooth sculptured surfaces (especially
marble) than for the natural building stone as presented
here. However, it has to be kept in mind that any surface
color of a building stone is darkened by the melanised
organisms. In contrast to the green color of algae (and
some bright colors of crustose lichens), this darkening is
not perceived as a ‘‘biogenic’’ stain, but rather as a natural
color of the stone or as a successive darkening by other
factors, e.g. soot deposits.
Melanization is an essential feature for protection
against high light intensities, ultraviolet and even ionizing
radiation (Bell and Wheeler 1986; Dadachova and Casa-
devall 2008. However, melanin is not essential for growth
in the dark and may be not expressed in isolates grown under
laboratory conditions (Fig. 5). Accordingly, in laboratory
cultures of Cladosporium isolates, just very thin cell walls
could be observed.
The isolation procedure of melanins from cell walls of
Phaeococcomyces illustrates the rigidity of the melan-
ised cell wall. Even boiling in diluted hydrochloride
solution over several hours did neither destroy the mel-
anised cell wall layer nor the molecule itself. All other
features of the cell, including all structures of the cell
envelope were destroyed (Fig. 8). On building stone,
after cell death and decay of all other organic com-
pounds, the melanised cell wall fragments remain on the
site for relatively long times. Since the filamentous fungi
are endolithic, these particles are not just attached to the
building surface, but are deposited in deeper layers of
the material.
Conclusion
Although colonization of building surfaces by microor-
ganisms and their contribution to biogenic weathering is a
well known fact, differences in the species composition or
in species diversity has been rarely addressed. The pre-
sented study shows a clear distinction between certain
specialists, just present in either of the both wall areas
under investigation and a low number of generalists.
Organisms from both groups may affect the building
material. Some lichens may take part in the formation of
large scales (cf. Fig. 3a) on sandstone substrata. Algae and
particularly black fungi contribute surface stains. It is
likely that exposition and moisture regime strongly influ-
ence the dominance of either of these groups, but does not
necessarily reduce or even exclude algal or fungal growth.
Thus, also intervention in moisture regimes on building
surfaces may change, but not necessarily reduce microbial
growth on building material.
Fig. 8 ‘‘Melanin ghosts’’ of Phaeococcomyces, transmission electron microscopy of ultrathin sections. a Cross section. b Sagittal section,
depicting the area of a former septum (asterisk)
Environ Earth Sci (2011) 63:1713–1722 1721
123
Acknowledgments Funding of this project by the DBU (Deutsche
Bundesstiftung Umwelt) is gratefully acknowledged.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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