ARTICLE IN PRESS

International Biodeterioration & Biodegradation 60 (2007) 50–59

www.elsevier.com/locate/ibiod

Fungal and algal biomass in biofilms on artificial surfaces quantified by

ergosterol and chlorophyll a as biomarkers
Solvig Görsa,, Rhena Schumanna, Norbert Häubnerb, Ulf Karstena
a
Department of Biological Sciences, Applied Ecology, University of Rostock, Albert-Einstein-StraX e 3, D-18051 Rostock, Germany
b
Department of Plant Ecology, Evolutionary Biology Centre (EBC), Uppsala University, Villavägen 14, SE-75236 Uppsala, Sweden
Received 26 May 2006; received in revised form 14 July 2006; accepted 5 October 2006
Available online 27 November 2006

Abstract

The occurrence of microbial colonisation on artificial surfaces in urban areas (building facades, roof tiles) and interiors (damp rooms:
silicone sealings, wallpaper) causes not only an aesthetically unacceptable discolouration of the surface, it also represents a conspicuous
problem in terms of biodeterioration, accelerated weathering and a potential health risk for humans by fungal mycotoxines. The most
conspicuous organisms responsible for biofouling terrestrial surfaces are fungi and green microalgae as well as cyanobacteria.
Microorganisms are often estimated by cultivating, but the biomass can be misjudged because of contaminations or the existence of non-
culturable cells and spores. By using ergosterol as a specific biomarker for living fungi and yeasts in combination with chlorophyll a for
aeroterrestrial microalgae it is possible to quantify fungal and algal infection independent from cultivation and undisturbed by surface
(dis)colouration. Using HPLC and photometric methods, conversion factors for microbial biomass were determined in representative
fungal and algal species: 5 mg ergosterol g1 fungal dry biomass and 23 mg chlorophyll a g1 algal dry biomass. The applied wipe
technique allowed a non-invasive sampling and evaluating of infections per square meter. Very low detection limits for ergosterol and
chlorophyll a permitted determining fungi and algae before they become macroscopically visible. Up to 36 mg ergosterol m2 and 180 mg
chlorophyll a m2 were detected on outdoor artificial surfaces, equivalent to approximately 7 g m2 fungal dry biomass and 8 g m2 algal
dry biomass. The distribution and the composition of the microbial communities varied strongly between the sampling locations. Fungi
were observed above windows of damp rooms or at more sun-exposed locations, whereas algae covered more wet and shadowed surfaces.
The established methods are well suited for the precise joint determination of fungal and algal biomass in microbial communities of
natural biofilms on artificial surfaces as a pre-condition for the development of prevention strategies against microbial colonisation.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Fungi; Microalgae; Facades; Quantification of fungal infection; Quantification of algal infection; Ergosterol; HPLC; Chlorophyll a; Extraction

1. Introduction contributes to the process of weathering, i.e. the destruc-

Fungi ubiquitously occur in the natural environment,
they colonise natural hard substrata such as soil, wood and
stone, as well as anthropogenic surfaces in urban areas
such as concrete, roof tiles, building facades or coated
metals. In addition, these organisms are also found in
many indoor localities such as wallpaper, wooden furniture
or silicone sealings, especially in damp rooms. The
occurrence of fungi on artificial surfaces constitutes not
only an unacceptable aesthetical problem, but also strongly
Corresponding author. Tel.: +49 381 498 6095: fax: +49 381 498 6072.
E-mail address: solvig.goers@uni-rostock.de (S. Görs).
tion of the infected material. The underlying mechanisms
are related to hydrolysis of organic material, e.g. cellulose
in wood, paper, paintings, plastics and rubber polymers
(e.g. Borel et al., 1982; Pommer and Lorenz, 1985), and the
production and excretion of organic acids (Gómez-Alarcón
et al., 1994). Indoor environments in particular are a
potential health risk for humans if fungal fouling is present
due to the formation of allergens and toxins (Jovanovic
et al., 2004), but also outdoors a lot of mould spores
are detectable. For example, in Germany on a warm
summer day more than 20,000 fungal spores m3 air
were measured in comparison to 100–2000 pollen grains
(Gravesen, 1979).

0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2006.10.003
 ARTICLE IN PRESS
S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59
While Alternaria, Aspergillus and Penicillium are wide-
spread fungal genera causing grey to black discolorations
on artificial surfaces outside (Brill, 1995), Cladosporium
and Penicillium were identified as the most abundant
fungal genera inside buildings (Dharmage et al., 1999). The
typical method of detecting fungal infection is to take a
smear or to collect an air sample followed by cultivation on
agar plates and the counting of colony forming units (cfu)
(e.g. Jovanovic et al., 2004). However, the results can be
misleading, i.e. false positive because of contaminations,
e.g. by air borne or spores from pot plant soil or by the
used swabs or plates as a failure of the technique. The
results can also be false negative because numerous
microorganisms are not culturable especially when they
originate directly from natural environments (Edwards,
2000). In addition, the cfu counting method is not suited
for quantification of fungal biomass because fast growing
species out-compete and hinder slower taxa (e.g. Bayrock
and Ingledew, 2004) and its inability to distinguish spores
from hyphal fragments of various lengths.
Fungi within mouldy houses are often characterised by
their odour that can be analysed by emitted volatile
substances, like geosmin. Such substances are collected by
adsorption on Tenax material from the respective compart-
ment air (Nilsson et al., 1996; Sunesson et al., 1996, 1997)
and used as a qualitative indicator for an infection. This
method is not applicable outdoors because these com-
pounds are rapidly scattered by wind. Fungi produce many
other volatile substances (alcohols, ketones, ethers, esters,
terpenoid compounds). However, the production is highly
dependent on both medium and species (Bjurman and
Kristensson, 1992; Sunesson et al., 1995). Therefore, these
compounds cannot be used to quantify the extent of a
fungal colonisation.
Ergosterol as a main component of fungal membranes
seems to be a proper biomarker for quantification of
biomass of these organisms, even though its content does
depend on various factors, such as culture age, growth rate,
carbon and nutrient availability, temperature and oxygen
(Hurst et al., 1997; Charcosset and Chauvet, 2001;
Dawson-Andoh, 2002). Nevertheless, in studies on differ-
ent fungal taxa, the average mycelial ergosterol concentra-
tion was found to be close to 5 mg g1 dry biomass (Hurst
et al., 1997). From that, a conversion factor equivalent to
200 mg fungal dry biomass mg1 ergosterol can be
calculated. Gessner and Chauvet (1993) suggested a similar
conversion factor of 182 mg fungal dry biomass mg1
ergosterol for aquatic hyphomycetes. Based on the
assumption that ergosterol is labile and undergoes a rapid
degradation upon cell death, a lot of environmental
microbiologists use this molecule as an indicator not for
total but exclusively for living fungal biomass (e.g. Newell
et al., 1987). In contrast to that, ergosterol was very stable
as a pure compound and when associated with dead fungi
with a decrease of o34% during 2 months when protected
from sunlight (Mille-Lindblom et al., 2004). Thus, the
estimation of ergosterol concentrations can be a promising
51
strategy to estimate living and possibly slightly degraded
fungal biomass on artificial surfaces.
So far, ergosterol was applied as a biomarker for fungal
biomass on natural substrates, e.g. in streams (Tank and
Webster, 1998; Tank et al., 1998) and on mouldy indoor
building materials (cf., Hippelein and Rügamer, 2004;
Pasanen et al., 1999). However, the two latter research
teams quantified ergosterol using gas chromatography and
mass spectrometry (GC/MS), which are precise but rather
time consuming methods. Nevertheless, Hippelein and
Rügamer (2004) confirmed the ergosterol concentration
on building materials as a suitable indicator for fungal
biomass in damp rooms. While at least some research has
been undertaken on the suitability of ergosterol as a fungal
biomarker for interior building materials (e.g. Szponar and
Larrson, 2000), there is one publication concerning
artificial outdoor surfaces (external wooden panels, Bjur-
man, 1999). Most of the data is not given in units per area
and does not provide an accurate assessment of infection
severity per square meter e.g. at building facades.
Quantitative results on concurrent fungal and algal
substrate colonisation are not available.
Therefore, the aim of the present study was to apply
ergosterol as a fungal biomarker for various in- and
outdoor artificial surfaces, e.g. building facades, fences,
containers, etc. Another goal was to establish a simple,
rapid, reliable and economical method of sampling,
extraction and HPLC for an area-based ergosterol
quantification. The magnitude of fungal biomass on
diverse materials is given. Since fungi occur together with
aeroterrestrial green algae on many artificial surfaces,
ergosterol and the algal pigment chlorophyll a (Schumann
et al., 2005) were measured on the same material to
compare the infection degree by both organism groups.
Because we never observed any cyanobacteria on sampled
facades and diatoms only rarely in our region, all detection
methods were optimised for chlorophytes (Schumann et al.,
2005; Eggert et al., 2006).
2. Materials and methods
2.1. Isolates and culture conditions
Fungal and yeast cultures (Table 1) were provided by the microbiology
working group of the University of Rostock. They were grown at room
temperature in darkness in sterile plastic Petri dishes on malt extract
peptone agar (Medium 90, DSMZ—German Collection of Microorgan-
isms and Cell Cultures, Braunschweig, Germany) for at least 3 weeks
(fungi) and 3 days (yeasts). All fungi with exception of the red yeast
Rhodotorula sp. (Basidiomycota) belong to the Ascomycota and were
identified by small subunit ribosomal DNA (18SrDNA) sequencing (cf.,
Berg et al., 2005; Nirenberg and Zachow, personal communications).
Strains were isolated from soil or rhizospheres of strawberries and
potatoes, but all genera are biofouling relevant, too (Brill, 1995).
Aeroterrestrial microalgae (Table 2) were grown according to
Schumann et al. (2005) in 250 ml glass flasks filled with a modified Bold’s
Basal medium (MBBM) (Starr and Zeikus, 1993) under the following
conditions: 20 1C, photon fluence density of 35–40 mmol photons m2 s1,
provided by daylight lamps (Osram L18W/19, Germany); light:dark cycle
of 16:8 h. Cultures were aerated with filtered air to keep the cells in
 ARTICLE IN PRESS
52 S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59

suspension and supplied with CO2. They were harvested after 7 days in
logarithmic phase (ROS 47/4, 55/3, 77/1) or 3 weeks in stationary phase
(ROS 7/4, 48/2, SAG 2007, 2040). All species belong to the Treboux-
iophyceae (Karsten et al., 2005; Eggert et al., 2006; Friedl, personal
communication) and were isolated from artificial surfaces in Germany
except SAG 2007, which is a soil isolate.
2.2. Sampling
For the quantification of co-occurring fungi and algae on artificial
surfaces, a survey of various locations was undertaken in July and August
2004 and 2005 (Table 3). Eight replicate samples of 4 cm2 area were taken
at each date and location. The individual biofilm samples were transferred
into 2 ml Eppendorf tubes containing 1 ml liquid medium. They were
wiped manually several times, at least threefold, in dependence of the
thickness of the biofilm and the roughness of the surface with a wet cotton
swab, which was rinsed in-between in this fluid. Medium and cotton swab,
containing both the sample, were further processed together. Four
replicates were kept in MBBM and darkness for the chlorophyll a
determination. Within 2 h, the microalgae were filtered onto Whatman
GF/F glass-fibre filters. Sterile filtered water was taken as the transport
medium for the four replicates of the ergosterol analysis. For the
quantification of indoor fungal colonisation, sampled wallpaper was cut
into 4 cm2 pieces or areas were wiped as described above in three replicates
because of a lower patchiness here. All sampled material was stored at
18 1C protected from light and UV radiation until chemical analyses for
o4 weeks for ergosterol determination and o1 day for chlorophyll a
quantification.
Table 1
Ergosterol content (mg g1 fungal dry biomass) of predominant repre-
sentative fungal species on artificial surfaces (n ¼ 3, mean7standard
deviation)
Fungal culture Isolate Ergosterol content
(mg g1 fungal dry
biomass)
2.3. Ergosterol extraction
Cultivated fungi (Table 1) and natural samples (Table 3) were cooled
down to 80 1C and lyophilised at a vacuum of 5  104 bar (Steris
LYOVAC GT2). Definite amounts of dried biomass of cultures or each
4 cm2 replicate sample were extracted by refluxing in 1 ml 100% methanol
(p.a.) in 15 ml PE tubes for 2 h at 70 1C in a water bath, respectively.
Subsequently, 1 ml of a methanolic KOH solution (0.2 mol l1) was added
for saponification of ergosterol esters by refluxing for another 30 min,
followed by two re-extractions with 1.5 ml n-pentane (p.a.) under rigorous
shaking by a Vortex. One ml aqua dest. was added to the extract which
was then centrifuged 2 min at 6200g in order to get a clearly defined phase
separation. Each non-polar phase was transferred into micro tubes and
evaporated under a nitrogen stream. Dried extracts were stored at 18 1C
protected from UV radiation till HPLC-quantification for less than
another 3 weeks. The extraction of ergosterol was almost complete (4
98%) up to a dried fungal biomass of around 5 mg (cultures) or an
infection of 30 g m2 surface, respectively.
2.4. HPLC separation of ergosterol
Dried extracts were dissolved in dependence on the expected ergosterol
content of the sample in 0.2–1 ml 100% methanol (p.a.) by sonification for
5 min. Insoluble substances were removed by centrifugation at 16,000g.
Ergosterol was quantified according to Newell et al. (1988) with an Agilent
HPLC-system (Series 1100) consisting of a degasser, quaternary pump,
autosampler, column thermostat and diode array detector. The separation
was carried out at 25 1C by injection of 20 ml sample onto a 250  4 mm
Hypersil-ODS-column (5 mm, Knauer) protected with 5  4 mm precol-
umn at a flow rate of 0.8 ml min1 with 100% methanol as the eluent
under isocratic conditions. The retention time of ergosterol was 9.1 min.
The peak area at the absorption maximum of 282 nm was used for
quantification with an external standard (Sigma, Germany) via a four-
point-calibration. The detection limit for ergosterol was 25 ng ml1 and
1.25 ng cm2 surface, respectively.

Alternaria alternata 1 PR 29-12-1 3.370.6 2.5. Chlorophyll a determination

Alternaria alternata 2 PR 16-12-1 7.170.5
Fusarium culmorum B 19-88-2 4.270.3 The frozen samples were extracted stepwise after mechanical cell
Fusarium graminearum B 19-88-1 3.670.2 disruption according to Schumann et al. (2005). The homogenates were
Paecilomyces carneus BR 4-2-6 5.970.4 incubated with 5 ml dimethyl formamide (DMF) p.a. (Porra et al., 1989) in
Paecilomyces BSB 1-2-1 6.170.3 15 ml polyethylene tubes for 24 h in darkness at 5 1C. All samples were
marquandii shaken occasionally with a Vortex. After centrifugation for 10 min at
Penicillium italicum BB 1-1-19 3.870.2 6200g, the chlorophyll a concentration of the supernatants was determined
Penicillium sacculum RE 4-2-1 4.370.7 photometrically. The pellets were re-suspended in 3 ml DMF and kept
Rhodotorula sp. BB 2-3-18 4.8–6.2a for another 24 h before another chlorophyll quantification was carried
Trichoderma viride 1 BSE 1-1-10 4.6–5.6a out. Because of the high microalgal biomass in the biofilms this
Trichoderma viride 2 RB 1-2-20 4.770.1 second extraction contributed mostly more than 20% to total chlorophyll
a concentration. Therefore, a third extraction with 3 ml DMF was
a
n ¼ 2. applied.

Table 2
Chlorophyll a content (mg g1 algal dry biomass) of representative strains of aeroterrestrial microalgae on artificial surfaces (n ¼ 3, mean7standard
deviation)

Algal culture Isolate Chlorophyll a content (mg g1 algal dry biomass)

‘‘Chlorella’’ angustoellipsoidea/trebouxioides ROS 7/4 26.670.5

‘‘Chlorella’’ angustoellipsoidea/trebouxioides ROS 48/2 21.370.3
‘‘Chlorella luteoviridis’’ ROS 77/1 26.674.8
Lobosphaera incisa SAG 2007 23.871.5
Pseudococcomyxa spec. SAG 2040 25.971.3
Stichococcus spec. ROS 47/4 identical to SAG 2059 17.374.1
Stichococcus spec. ROS 55/3 identical to SAG 2060 21.676.8

ROS: culture collection of the University of Rostock, SAG: culture collection of algae at the University of Göttingen, Germany.
 ARTICLE IN PRESS
S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59 53

Table 3
Sample locations, surface characteristics and sample code of investigated outdoor and indoor artificial substrata

Sample locations outside Surface characteristics Sample code

Garden fence stake, Rostock, Beim Pulverturm Concrete, uncoated, sun-exposed, partly C1
lichens
Shopping centre, Bützow, Nebelring Concrete base, uncoated, sun-exposed, partly C2
lichens
Garden wall, Rostock, Bei der Tweel 2 Concrete wall, uncoated, shaded C3
Stairs, waste disposal court, Rostock, Albert-Einstein-StraXe Concrete, uncoated, shaded C4
Railway bridge, Rostock, Erich-Schlesinger-StraXe, eastern side Concrete wall, uncoated C5
Railway bridge, Rostock, Erich-Schlesinger-StraXe, western side Concrete wall, uncoated C6
Balcony, Rostock, Brahe-StraXe Plastics P
Facade, Rostock, Südring 46, south-eastern side Thermal insulated combined system, dark F1
yellow coated
Carport, Lieblingshof Plaster, uncoated, shaded F2
Facade, Rostock, Südring 22, north-western side Thermal insulated combined system, light F3
yellow coated
Cellar, Lieblingshof Plaster, uncoated, shaded F4
Roof, Lieblingshof Roof tiles, partly lichens R
Laundry rack, Rostock, Südstadt White coated metal CM1
Switching box, Rostock, Jungius-StraXe Grey coated metal, shaded CM2
Switching box, Rostock, Max-Planck-StraXe Grey coated metal CM3
Wooden bench, Rostock, Brahe-StraXe Uncoated wood W
Recycling container for white glass, Bützow, Nebelring White coated compressed wood CW1
Recycling container for green glass, Bützow, Nebelring Green coated compressed wood CW2
Wooden bench, Rostock, near city hall White coated wood CW3
Recycling container for brown glass, Bützow, Nebelring Brown coated compressed wood CW4
Sample locations inside Surface characteristics Sample code

Cellar room, water damage, grey discolouration, single occupancy White painted woodchip wallpaper, detached Wallpaper grey
house, Rugensee
Cellar room, water damage, grey discolouration, single occupancy White painted woodchip wallpaper, wiped Wipe sample grey
house, Rugensee
Cellar room, water damage, dark grey discolouration, single White painted woodchip wallpaper, detached Wallpaper dark grey
occupancy house, Rugensee
Cellar room, water damage, black discolouration, single occupancy White painted woodchip wallpaper, detached Wallpaper black
house, Rugensee
Unaired living room, apartment building, Rostock-Dierkow Silicone sealing on windows, wiped Silicone sealing
Bathroom, single occupancy house, Schoenebeck Structure wallpaper, detached Wallpaper bathroom
Bedroom, single occupancy house, Badendiek Structure wallpaper, detached Wallpaper bedroom

All samples were taken in Rostock and some nearby villages.

2.6. Statistics
Means and standard deviations were calculated for replicate measure-
ments as well as averaged biomarker contents in microorganisms (medians
for comparison) and medians were estimated for average infections of
different types of material or sample locations. Tests of normal
distribution of data were done with SigmaStat 2.03. Tests to show
significance were not done because of high natural standard deviations of
microbial biomasses caused by their patchiness on the artificial surfaces.
3. Results
biomass (median 4.7) (Table 1). The mean value can be
used for the conversion into fungal biomass because the
data were normally distributed. Therefore, by calculating
the reciprocal value of the mean, a conversion factor of
206 mg fungal dry biomass mg1 ergosterol was defined.
Aeroterrestrial green algae exhibited chlorophyll a
contents between 17.3 and 26.6 mg g1 dry mass resulting
in a mean value of 23.373.4 mg g1 dry mass (median 23.8)
(Table 2). A conversion factor of 43 mg algal dry biomass
mg1 chlorophyll a was derived from the mean.

3.1. Biomass specific ergosterol content in fungal cultures
and chlorophyll a concentrations in aeroterrestrial
microalgae
The ergosterol concentration based on fungal dry
biomass varied in the investigated culture fungi only
between 3.3 and 7.1 mg g1 dry biomass resulting in an
average value of all measurements of 4.971.2 mg g1 dry
3.2. Ergosterol and chlorophyll a concentrations in natural
biofilms on artificial surfaces
The measured ergosterol amounts on artificial surfaces
ranged from 0.15 to 36.0 mg m2. Average concentrations
indicating material-bound fungal infections were 6.42
(3.52–7.30) mg m2 on coated metal, 5.52 (0.28–30.9)
mg m2 on concrete, 3.96 (1.49–14.7) mg m2 on coated
 ARTICLE IN PRESS
54 S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59

wood and 0.62 (0.22–4.41) mg m2 on facades. In compar- pigment was almost undetectable (Fig. 2, Table 3). The
ison for dependences from sun-exposition, lichen-free determined chlorophyll a concentrations ranged from 0.06
strongly sun-exposed locations showed an average infec- to 180 mg m2. Average concentrations indicating materi-
tion of 5.35 (1.49–14.7) mg ergosterol m2 and extremely al-dependent algal infections were 79.0 (0.06–125) mg m2
shaded places 3.21 (0.22–7.84) mg ergosterol m2. The on facades, 51.2 (21.0–130) mg m2 on concrete and 0.49
lowest values were found on untreated concrete and plaster (0.13–50.9) mg m2 on coated wood. In comparison for
in shaded locations, the highest contents on an untreated dependences from sun-exposition, lichen-free strongly sun-
fence stake partly covered with lichens, as well as a painted exposed locations showed an average infection of 0.18
compressed wood surface, both were in sun-exposed (0.06–0.77) mg chlorophyll a m2, in contrast to extremely
surfaces (Fig. 1, Table 3). shaded with 125 (99–130) mg chlorophyll a m2.
In contrast to ergosterol, north-western sides of facades The standard deviations for the determination of
or other shaded locations showed high concentrations of chlorophyll a and ergosterol, and algae and fungi,
chlorophyll a, while on strongly sun-exposed surfaces this respectively, were high with 11–60% (fungi) and 9–46%

15 30.9
Ergosterol (mg m-2)

10

0
1
2
3
4
5
6
P
F1
F2
F3
F4

1
2
3
W

1
2
3
4
C
C
C
C
C
C

M
M
M

W
W
W
W
C
C
C

C
C
C
C
concrete plastics facades roof tiles coated metal wood coated wood

Fig. 1. Ergosterol content (mg m2) in natural biofilms on outdoor artificial surfaces (n ¼ 4, mean7standard deviation, sampling stations: see Table 1).

150
Chlorophyll a (mg m-2)

100

50

0
3

F4

F2

F3

F1

2
W

4
C

W
C

concrete facades roof tiles coated metal wood coated wood

Fig. 2. Chlorophyll a content (mg m2) in natural biofilms on outdoor artificial surfaces (n ¼ 4, mean7standard deviation, sampling stations: see Table
1).
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S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59 55

Fungal dry biomass (g m-2)

I
2

II

III
0

0 2 4 6
Algal dry biomass (g m-2)

Fig. 3. Correlation analysis of calculated fungal and algal dry biomass (g m2) in natural biofilms on outdoor artificial surfaces (n ¼ 4, mean7standard
deviation, conversion factors: 206 mg fungal dry biomass mg1 ergosterol, 43 mg algal dry biomass mg1 chlorophyll a).

Table 4
Ergosterol and chlorophyll a concentration (mg m2) and the respective fungal and algal dry biomass (g m2) of lichens on an artificial surface (switching
box, coated compressed wood) (n ¼ 4, mean7standard deviation, conversion factors: 206 mg fungal dry biomass mg1 ergosterol, 43 mg algal dry
biomass mg1 chlorophyll a)

Sample location Surface Lichen Ergosterol Chlorophyll Fungal dry Algal dry Chlorophyll Fungi/algae
characteristics (mg m2) a (mg m2) biomass biomass a/Ergosterol ratio (dry
(g m2) (g m2) ratio (g g1) biomass)

Switching box Grey coated Xanthoria sp. 127732 253747 26.176.6 10.972.0 1.99 2.39
Bützow compressed wood
Schwaaner StraXe
yellow lichen
Switching box Grey coated Parmelia sp. 135741 3007113 27.878.4 12.974.9 2.23 2.16
Bützow compressed wood
Schwaaner StraXe
grey–green lichen

(algae) of the means. They were extremely high for fungi on
some surfaces, a roof (159%), a recycling container (87%),
a switching box (72%) and a cellar wall (77%). The
patchiness of fungi was stronger than that of algae, which
showed particularly high patchiness only if the values of
chlorophyll a were low (76% and 83% on two occasions).
The irregular spreading of the microorganisms was
macroscopically visible but the quantitative extent of this
fact was underestimated by eye.
3.3. Correlations between fungal and algal dry biomass on
artificial surfaces
biomasses accumulated. Both microorganism groups co-
occurred almost in equal parts in cluster II, which included
very different materials (concrete, wood, paint, roof tiles)
predominantly on shaded surfaces. The same materials and
exposition conditions were favoured also by algae alone
(cluster III).
Lichens, which covered an always sun-exposed, grey
coated switching box of compressed wood, were hotspots
of both algal and fungal biomass. Fungi dominated the
total dry biomass in the lichen samples (Table 4). The
lichens were identified as Xanthoria spec. and Parmelia
spec. (Moberg and Holmåsen, 1992).

From ergosterol and chlorophyll a as biomarkers for
fungi and microalgae, the respective microorganisms’
biomasses were estimated. Some artificial surfaces were
colonised almost only by fungi or algae (cluster I and III),
while on others fungi and algae occurred to the same
degree (cluster II) (Fig. 3). In cluster I, all strongly sun-
exposed surfaces with high fungal and very low algal
3.4. Ergosterol concentration on walls inside buildings
The ergosterol concentrations on the indoor walls
investigated, ranged between 4.44 and 29.8 mg m2
(Fig. 4). A value of 29.8 mg m2 is considered to be high
and was measured in one of the three cellar rooms after
water damage. The estimated fungal dry biomass varied
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56 S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59

30
6

Fungal dry biomass (g m-2)

Ergosterol (mg m-2) 20 4

10 2

0 0

k
y

m
ac
e

in

oo

oo
gr

gr

gr

al
bl

hr

dr
se
er

k
e

er
r
pl

be
ap

da

ba
e
ap
m

on
lp

er
sa

er

er
lp
al

lic

ap
ap

ap
al
e
w

si

lp
w
ip

lp

lp

al
w

al

al

w
w

w
water damage

Fig. 4. Ergosterol concentration (mg m ) and calculated fungal dry biomass (g m2) in biofilms on inner walls after water damage and in damp rooms
2

(n ¼ 3, mean7standard deviation, conversion factor: 206 mg fungal dry biomass mg1 ergosterol).

from 0.9 to 6.1 g m2 (Fig. 4). A visual gradient in programs are necessary. The lower detection limit for fungi
discolouration from grey to black was correlated to was 12.5 mg ergosterol m2 (equivalent to 2.6 mg fungal dry
increasing fungal biomass. The amount of fungi on a biomass m2). Thus, fungi were detectable before they were
silicone sealing at a window of an unaired living room in macroscopically visible or their odour was noticeable.
winter was with 4.771.0 g m2 almost as high as the While the extraction was nearly complete and reproducible
darkest decay zone in the cellar. The fungal infections of (cf., Gessner et al., 1991), the precision in respect to the
wallpaper in damp rooms ranged with 1.770.3 (bathroom) sampled areas was dependent on the investigator. How-
and 3.571.9 g m2 (bedroom) near the values in the cellar ever, the main cause of high standard deviations of biomass
rooms determined after the water damage. per area was the patchy distribution of the fungi on the
artificial surfaces (cf., Hippelein and Rügamer, 2004) and
4. Discussion microalgae. A direct evaluation of ergosterol concentration
against cfu for building materials of inner walls was done
4.1. Methodological considerations by Gutarowska and Zakowska (2002). The dependence
was presented as an exponential function, but there were
In contrast to former investigations on fungal colonisa- positive or negative differences between predicted and
tions on building material with dry matter as reference actually culturable cells. This may be attributed mainly to
parameter (Hippelein and Rügamer, 2004; Pasanen et al., problems of this culture dependent method and should be
1999), the sampling in the present study was related to an re-evaluated by direct cell counts.
area basis. That is comparable with the correlations Methodical considerations concerning algal biomass
between ergosterol content and cfu on indoor walls estimations are discussed in Schumann et al. (2005) and
(Gutarowska and Zakowska, 2002) and unhampered by Eggert et al. (2006).
the materials specific weight or sampling depth. The
applied wipe technique allowed an almost complete 4.2. Applicability of ergosterol as a biomarker for fungi on
harvesting biomass, estimated by eye, without any destruc- facades
tion of the surface. This biomass calculation based on area
seems to be more appropriate to assess the spreading of Contaminations and spores as well as non-culturable
biofilms on artificial surfaces, e.g. walls, wallpaper, than a cells can lead to a misjudgement of fungal infections if
relation to infected material weight. However, the relation determined by wiping from surfaces and cultivating
to material is of course also possible if needed. colonies. The formation of volatile substances in fungi is
The applied HPLC method for ergosterol determinations influenced strongly by environmental conditions and these
was modified after Newell et al. (1988) and is more time compounds are not easy to detect outdoors. Thus, a
and cost efficient than GC/MS methods (cf., Hippelein and biomarker like ergosterol is more appropriate for calcula-
Rügamer, 2004; Pasanen et al., 1999), because no tion of fungal biomass, determined best via a low cost and
derivatisation procedure and no time controlled gradient fast HPLC method. An application of a colour scale such
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S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59
as the one for algae (cf., Donner et al., 2002) seems to be of
little use, too, because fungi have different colours or are
colourless. A grey scale for black fungi would be possible
but only indoors because there the colour is not masked by
algal pigments and dust.
Ergosterol concentrations vary among different fungal
species, among isolates of the same species, and even within
a strain depending on the physiological state. A number of
factors, including age, growth rate, carbon and nutrient
availability, temperature and oxygen, have been found to
affect mycelial ergosterol contents (Dawson-Andoh, 2002;
Hurst et al., 1997). While pollutants like heavy metals
reduce the metabolic activity in fungi without affecting the
ergosterol content, a treatment with the fungicide Zineb
decreased the ergosterol content in the dry biomass to 57%
compared to the control (Barajas-Aceves et al., 2002).
Thus, the colonisation of surfaces treated by Zineb or other
ergosterol reducing agents will be underestimated by the
ergosterol method.
Many authors described ergosterol as a quantitatively
important constituent of fungal cells and determined a
concentration of ca. 5 mg g1 fungal dry biomass (Hurst et
al., 1997). However, the ergosterol content differed
between species and even isolates. Therefore, the conver-
sion factor into dry biomass was evaluated for fungi
representative for artificial surfaces (Table 1). The range of
ergosterol contents estimated in this study was well within
the range of other studies, but more on the lower range
(Gessner and Chauvet, 1993: 2.3–11.5 mg g1; Pasanen
et al., 1999: 2.6–37 mg g1; Barajas-Aceves et al., 2002:
2.38–13.06 mg g1; Dawson-Andoh, 2002: 0.43–3.66
mg g1). Moreover, the values had a much lower variability
compared to the literature. Therefore, the ergosterol
concentration is a suitable indicator to estimate fungal
biomass in microbial infections on various outdoor
artificial surfaces and indoor walls. The average conversion
factor amounted to 206 mg fungal dry biomass mg1
ergosterol.
4.3. Comparison of microbial infection on different surfaces
A determination of mould growth in damp rooms on
plaster, paint and wallpaper was done by Hippelein and
Rügamer (2004) by GC/MS. The concentrations of
ergosterol ranged from 0.1 to 130 mg g1 dry mass and
depended on the carbon content of the infected material, so
that the ergosterol content was highest with up to 84 mg g1
dry mass on light wood-chip wallpaper. In comparison,
indoor dust measurements in a bedroom floor resulted in
3.8 mg ergosterol g1 of dust (Dharmage et al., 1999). An
ergosterol content of 1 mg m2 was equal to 4.8 mg g1 dry
mass in this study for the wood-chip wallpaper taken in a
cellar after a water damage as well as for the structured
wallpaper from a bathroom. The estimated ergosterol
values equalled 21.3 and 143 mg g1 dry mass, respectively.
These concentrations are much higher than those reported
by Hippelein and Rügamer (2004), but in contrast to this
57
reference the mould on all sample locations was clearly
visible as grey to black patches. Wood chip insulation,
gypsum board and glass wool insulation of three buildings
with acute or previous moisture damage contained
ergosterol amounts from 0.017 to 65 mg g1 dry mass
(Pasanen et al., 1999). The highest values were determined
in organic (wood chip insulation, mean 13 mg g1), the
lowest in inorganic material (gypsum board, mean 0.20 mg
g1), indicating that mould growth is dependent on the
type of the infected material. The indoor infections
analysed in this study had to be assessed as high, because
the mould was always visible. Such strong fungal biofilms
can contain a high amount of mycotoxines and, therefore,
they hold a potential health risk.
However, fungal growth was less dependent on the type
of infected material on the outdoor artificial surfaces.
Direct or indirect sun radiation, the moisture of the surface
and the co-occurrence of aeroterrestrial green algae seem to
affect fungal growth. On the same building material a
much higher contribution of fungi to the total microbial
colonisation was determined at sun-exposed, more dry
south-eastern sides in comparison to more algal growth at
shaded, moister north-western sides. This observation
confirms the preferential occurrence of aeroterrestrial algae
on sun-protected building facades (Schumann et al., 2005).
Results of Bjurman (1999) on external wooden panels
showed also that exposed surfaces to the south were
colonised mainly by fungi. The higher fungal biomass on
sun-exposed surfaces can be explained by a higher
radiation and desiccation tolerance of many fungi com-
pared to other microorganisms (Atlas and Bartha, 1998).
This tolerance is supported by the production and
accumulation of UV-absorbing mycosporines and mela-
nins, which are typical sun screen metabolites in terrestrial
fungi, as well as antioxidants and by a compact tissue-like
colony organisation formed by thermodynamically optimal
round cells embedded in extracellular polymeric substances
(Gorbushina, 2003; Gorbushina et al., 2003).
In many cases, aeroterrestrial microalgae seem to appear
first on mineral surfaces, if they are sufficiently supplied by
nutrients (e.g. from rain-water), are rather moist by
condensation or rain and protected by direct sun radiation.
These algae need 100% humidity for their growth; many
species are even dependent at least temporarily on liquid
water (Schumann et al., 2004; Häubner et al., 2006). These
primary producers generate the organic basis (lysed cells,
exudates, etc.) for fungal growth on surface which would
otherwise not be infected heavily due to substrate limita-
tion.
In conclusion, the applied method for ergosterol analysis
is very well suited for the calculation of the fungal fraction
in microbial communities of natural biofilms on artificial
surfaces. Further investigations are necessary to explain,
how the intensity and composition of microbial infections
on building facades are influenced by the kind of affected
material and by environmental factors. Such information
would be the first step in the development of efficient and
 ARTICLE IN PRESS
58 S. Görs et al. / International Biodeterioration & Biodegradation 60 (2007) 50–59
ecologically sustainable strategies to prevent microbial
colonisation of building facades.
Acknowledgements
S.G. thanks the Governmental Department of Educa-
tion, Science and Culture of Mecklenburg-Vorpommern,
Germany, for a postdoc fellowship within the university
scientific program ‘‘Advancement of equal opportunity of
women in research and teaching’’. Financial support of the
German Research Foundation (DFG) through the project
KA899/13-1 is gratefully acknowledged. We thank T.
Friedl with his group for rDNA sequence analyses of our
algal strains, S. Göbel for her assistance in ergosterol
analysis, S. Lembcke for cultivation of algae and G. Ballin,
J. Lottmann, C. Zachow, H. Goschke (Microbiology,
University of Rostock) for providing of fungi as well as
H.I. Nirenberg (Federal Biological Research Centre for
Agriculture and Forestry, BBA) and C. Zachow for their
genetic identification. Many thanks to N. Cox who made a
check on the English language.
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