Original Paper

Indoor and BuiltEnvironment Indoor Built Environ 2004;13:63–74 Accepted: May 7, 2003

Airborne Fungi andActinomycetesConcentrations in the Air ofEskisehir City (Turkey)

Ahmet Asana Semra Ilhanb Burhan Sena

Ismuhan Potoglu Erkarab Cansu Filikb Ahmet Cabukb

Rasime Demirelb Mevlut Turec Suzan Sarica Oktena

Suleyman Tokurb

aTrakya University, Faculty of Arts and Sciences, Department of Biology, TR-22030 Edirne, TurkeybOsmangazi University, Faculty of Arts and Sciences, Department of Biology, TR-26480Meselik, TurkeycTrakya University, Medical Faculty, Department of Biostatics, TR-22030 Erdine, Turkey

Key Words

Fungi E Actinomycetes E Urban air E Biomass E

Fungal and Actinomycetes distribution E Bioaerosols

E Airspora

AbstractThe present study investigated the isolation and iden-

tification of airborne fungi from three different urban

stations located in Eskisehir (Turkey). Air samples were

taken by exposing a Petri dish with Rose-Bengal

streptomycin agar medium for 15min and after incuba-

tion the number of growing colonies was counted. The

sampling procedure for fungi was performed 35 times

at the research stations weekly between March and

November 2001. A total of 2518 fungal and 465

actinomycetes colonies were counted on 420 Petri

plates over a nine-month period. In total, some 20

mould species belonging to 12 genera were isolated.

Alternaria alternata, Cladosporium cladosporioides and

Scopulariopsis brevicaulis were the most abundant

species in the study area (13.66, 5.80 and 5.50% of the

total, respectively). Relationships between fungal spore

numbers, aerosol air pollutants (that is the particulate

matter in the air) and sulphur dioxide together with the

meteorological conditions were examined using statis-

tical analysis. Number of fungi and actinomycetes were

tested by multivariate analysis (MANOVA) according to

the areas and months. Fungal numbers were non-

significant according to the areas and months (p>0.05),

but the number of actinomycetes recorded was sig-

nificant (p<0.01).

Introduction

Living particles such as fungal propagules are ubiqui-

tous in the atmosphere. Fungal spores have even been

detected in the air of Signy Island in the maritime

Antarctic [1]. Fungal spores and other airborne structures

are found both indoors and out. Madelin [2], explained

that fungal aerosols are important in the spread of plant,

animal and human disease. Airborne fungi may cause

allergy in humans [3,4], so they are important from this

� 2004 Sage PublicationsDOI: 10.1177/1420326X04033843Accessible online at:www.sagepublications.com

Prof. Ahmet AsanTrakya University, Faculty of Arts and Sciences, Department of BiologyTR-22030 Edirne, TurkeyTel. þ90 284 2356405, Fax þ90 284 2354010, E-Mail ahmasan@hotmail.com

point of view. Some species of fungi such as Cladosporium

cladosporioides, C. herbarum, Penicillium brevicompactum,

P. chrysogenum,Aspergillus candidus,A. niger,A. versicolor,

etc. can provoke extreme allergic reactions in humans [5].

According to Singh [6], mould growth may contribute

to the sick-building syndrome as well as to allergy and

other environmental health problems. Furthermore,

airborne fungi cause spoilage of foods and are responsible

for many adverse health effects; the mycotoxins which they

produce may affect human and animals, and fungal

propagules can serve as an infective agent of plant disease.

In addition, these bioaerosols may cause eye and sinus

irritation, sore throat, headache, fatigue and dizziness [7].

The concentration of airborne micro-organisms are linked

to the level of airborne dust and various human activities.

In addition, the concentration of fungal spores in the air is

linked to both geographical region and seasonal variations,

meaning that it is dependent on wind, humidity, tempera-

ture, rainfall, altitude, vegetation and some specific

reservoirs of contamination [8–10]. Also, the air velocity

above any surface contaminated with moulds, the texture

of that surface, and vibration or other movement of the

contaminated material, will affect the number of fungal

spores released to the atmosphere [11]. Overall, the

prevalence of airborne fungi is highly variable and

determined by many factors.

Nocard (see [12]) recognised the pathogenic potential

of actinomycetes for the first time in 1888 and since then,

several aerobic actinomycetes have been a major source

of materials for the commercial drug industry, some of

which have proved to be useful for the production of new

antimicrobial agents [12]. Reponen et al. [13] noted that

airborne actinomycetes spores are important contaminants

in occupational and residential environments and the

spores of several actinomycetes species have been related

to the occurrence of allergic alveolitis and other health

effects.

There have been very few studies on airborne fungi in

Turkish cities. The studies that have been carried out have

monitored fungi in only a handful of cities including

Istanbul [14], Edirne [15–17], Bursa [18], Ankara [19]

and Izmir [20]. Harmanci et al. [21] determined that

Cladosporium and Aspergillus spores caused allergic reac-

tions in adult patients with asthma and/or rhinitis in

Eskisehir; they also explained that Cladosporium was the

commonest cause. The first study on airborne fungi in

Eskisehir was done by Atik and Tamer in 1994 [22]. These

workers also studied airborne bacteria in this city [23].

However, the city is developing and expanding year by

year and the population and industrial activities are

increasing. As a consequence the earlier work is now out

of date with the airborne fungal and actinomycetes

concentrations effectively unknown. So, the objective

of the present study was the determination of fungal

and actinomycetes concentrations in the different study

areas of the present city of Eskisehir. In particular, so far

as we can ascertain, studies of airborne actinomycetes have

not been made in Turkey although studies have been

carried out in other parts of the world (for example

[13,24]).

The rationale behind the work was that since actino-

mycetes and many fungi are potential allergens the study

may provide the basis for future work on health effects.

Because of the paucity of information of this type from

Turkey, the study has also increased our knowledge of

the distribution of fungal and actinomycetes flora in our

country. Mould allergy is common in Eskisehir city. The

official number of patients who applied to the Osmangazi

University Hospital at Eskisehir for mould allergy tests

are: 948 in 1999, 1185 in 2000, and 1053 in 2001 (Source:

Osmangazi University Hospital records) (Allergens from

several fungi were included in the mould tests: Penicillium

notatum, Cladosporium herbarum, Aspergillus fumigatus

and Alternaria alternata). As an aside, we did have the

expectation that we would record for the first time fungal

species new to Turkey and, perhaps, the world, but this

was not to be.

Materials and Methods

Sampling Sites

The salient features of the research stations, the number

of samples and other relevant information is given in

Table 1.

The concentration of airborne fungal spores and

actinomycetes from three urban areas in Eskisehir City

(1. Osmangazi University Meselik Campus; 2. Anadolu

University Yunusemre Campus; 3. Anadolu University Iki

Eylul Campus) (Figure 1 and Table 1) have been

measured. The first station has rich flora; there are 240

plant species and 41 varieties. Asteraceae and Fabaceae

families are very common. There are 363 plant species at

the second station where the Asteraceae, Fabaceae and

Lamiaceae families are common. The third station is in a

residential area, but some plants species belonging to the

Poaceae, Asteraceae and Fabaceae families are common

[25,26]. The first and second stations are in the north of the

city, but the third station is in the south. Distances

64 Indoor Built Environ 2004;13:63–74 Asan et al.

Fig. 1. A map of Eskisehir city (Turkey) map showing Eskisehir province (Top, left). The research stations are: 1. Osmangazi UniversityMeselik Campus; 2. Anadolu University Yunusemre Campus; 3. Anadolu University Iki Eylul Campus.

Table 1. Some features of the selected research stations and number of the samples taken

Samplingstation#

Number ofsample setstaken

Number ofpetri dishes ateach sampling*

Total number ofpetri dishes

Some features of thesampling station

1 35 4 140 Exposed to wind, neither mountainous nor natural forest (exceptfor occasional plantations). There are residential areas to the eastand west of the station. Latitude: 39� 440N, Longitude: 30� 290E,Altitude: 845m. Flora: plants belonging to Asteraceae andFabaceae families are common, followed by Brassicaceae,Lamiaceae, Boraginaceae, Poaceae and Rosaceae (14.7, 9.6, 8.5,6.8, 5.1, 4.2 and 1.9% respectively) [26].

2 35 4 140 Exposed to wind, thicketed area, few plantations or natural trees,residential area to the west of the station. The Centrum is to theeast of the station. Latitude: 39� 470N, Longitude: 30� 290E,Altitude: 819m. Some plants belonging to Asteraceae (9.9%),Fabaceae (7.9%) and Lamiaceae (6.0%) [26].

3 35 4 140 Exposed to wind, no mountains, natural forest, agricultural orrecreation areas. There is a meat processing plant to the east of thestation, some stockbreeding by peasant farmers. Latitude: 39�

480N, Longitude: 30� 320E, Altitude: 789m. The common plantfamilies are Poaceae, Asteraceae and Fabaceae. This station is at aspecial building lot on the University Campus.

Total 105 4 420

#1. Osmangazi University Meselik Campus; 2. Anadolu University Yunusemre Campus; 3. Anadolu University Iki Eylul Campus,

*2 for fungi and 2 for actinomycetes.

Fungi and Actinomycetes in Eskisehir City Indoor Built Environ 2004;13:63–74 65

between the research stations and a meteorological station

were measured by GARMIN GPS 12 CX device (Global

Positioning System; Made in Taiwan, under USA patent).

This device was also used to measure the altitude of the

research stations.

Sampling and Isolation Methods

The samples were taken in the morning (10.00–12.00) at

1.50m above the ground level. The sampling time was

always at the same time of the day. Concentration of

airborne fungi and actinomycetes were conducted over a

period of nine months at different sites (from March to

November 2001). Sampling was performed at one-week

intervals; but we did not sample on rainy days; thus,

counts of airborne microfungi and actinomycetes were

conducted only on days when the weather was stable and

dry. At the end sampling had been carried out for 35 weeks

(of 39) over the nine-month period.

The method used for the isolation of fungi and

actinomycetes was either the Petri plate gravitational

method or the Settle plate method [27,28]. Rose Bengal

streptomycin agar medium was used for isolation of the

fungi while Czapek’s solution agar (CZ) (Merck,

Germany) with cycloheximide (50 mg �mL�1) was used

for actinomycetes. The agar medium for the fungi was

made from: Dextrose 10 g, peptone 5 g, KH2PO4 1 g,

MgSO4 � 7H2O 0.5 g, Agar agar 15 g, sterile pure water

1000mL and used according to the following procedure:

1 g powdered streptomycin (Deva Inc., Turkey) was

dissolved in 33mL sterile pure water; 2mL of this solution

was added to 1000mL of the medium; and then 10mL of

a solution of 0.5 g powdered Rose Bengal stain (Sigma

Chemical Co., USA) dissolved in 150mL sterile pure

water was added. Two Petri dishes were used for each

sampling. After incubation at 27� 1�C, the concentrations

of airborne fungi and actinomycetes were calculated as

CFU (Colony forming units) (CFU/plate/15min).

Each colony of fungi was inoculated onto malt extract

agar (MEA) (Merck, Germany), CZ and potato dextrose

agar (PDA) (Difco, USA) media for identification and

incubated at room temperature (27� 1�C) for a period of

seven days after which colony diameters were measured.

Petri plates were first examined under the dissecting

microscope (a stereomicroscope) and then under a high

resolution light microscope to determine the colonial

features and the morphological structures of the fungi. The

determination of the morphological structures was carried

out on material mounted in a modified mounting medium,

Lacto-Cotton Blue, as proposed by Sime et al. [29].

Identification

Many different culture media were used for the iden-

tification of species. Fungal species were identified based

on micro- and macro-morphology, reverse and surface

colouration of colonies grown on CZ, MEA and PDA

media. Fungi were identified to genus level using Barnett

and Hunter’s work [30]. Cultures were identified to species

level (except actinomycetes) according to various mycolog-

ical references as below: Pitt [31] was followed for the

identification of Penicillium species. These species were

grown on three different media all prepared according to

the recipes of Pitt [31]. So, Czapek Yeast Extract agar

(CYA), MEA, and 25% glycerol nitrate agar (G25N) were

used for cultivation of Penicillium species and prepared

according to Pitt [31]. Each Penicillium culture was

inoculated in triplicate onto each medium and incubated

at three different temperatures (5, 25 and 37�C) for a

period of seven days in the dark. The works of Raper and

Fennell [32] and Klich [33] were used for identification of

Aspergillus species. So, Czapek Yeast Agar with 20%

sucrose (CY20S) and Czapek Yeast Agar (CYA25,

CYA37) were used to cultivate some Aspergillus species

and prepared according to Klich [33]. We also prepared

Czapek concentrate [31,33] from NaNO3 (30 g), KCl (5 g),

MgSO4�7H2O (5 g), FeSO4 � 7H2O (0.1 g), ZnSO4 � 7H2O

(0.1 g), CuSO4�5H2O (0.05 g) dissolved in 100mL distilled

water for addition to CYA, G25N, CYA25 and CYA37

media. An aliquot portion of 15mL of media was poured

into the Petri dishes as a standard procedure. The volume

of medium is important since depth of medium or head

space differences can lead to morphological changes

[33,34]. The Cladosporium and Alternaria sp. conidia and

other structures present on the microscope slides were

identified according to the descriptions of Ellis [35] and

Ellis and Ellis [36]. Fusarium sp. was identified following

the concepts of Nelson et al. [37]. Scopulariopsis brevicaulis

and Trichotecium roseum were identified according to

Hasenekoglu [38]; Ellis [35] was used for identification of

Ulocladium tuberculatum, Wardomyces ovalis and Torula

sp. Actinomycetes species could not be identified at the

genus or species level and only numerical results are

presented. Each colony of actinomycetes was inoculated

onto Tryptone Yeast Extract Agar (TYEA) prepared

from: tryptone (0.5 g), yeast extract (0.3 g), agar agar

(1.5 g) and sterilised and distilled pure water (100mL)

and incubated for a period of seven days at 27� 1�C.

Macroscopic and microscopic investigation of actinomy-

cetes was carried out according to Schall [39,40] and

Waksman [41].

66 Indoor Built Environ 2004;13:63–74 Asan et al.

All isolates identified in our study were deposited

in the Culture Collection at the Osmangazi University,

Department of Biology at the Eskisehir city (Turkey).

Citation of the author names presented in this paper

are standardised according to the Authors of Fungal

Names [42] (New online version of this revised book

can be obtained from: http://www.indexfungorum.org/

AuthorsOfFungalNames.htm). The list of accepted species

and synonyms in the family Trichocomaceae [43] was fol-

lowed for acceptable names of Penicillium and Aspergillus

species.

Climatological and Partial Air Pollutants Data

Weather data (monthly average temperature, monthly

total rainfall, monthly average relative humidity, monthly

average wind velocity and average monthly sunny times

(Table 3) had been recorded and was obtained from the

Directorate of Eskisehir Meteorological Office which is

positioned 9.2 km from the first station, 3.9 km from the

second station, and 0.5 km from the third station. Some air

pollutants including sulphur dioxide (SO2) (mg �m�3) and

respirablesuspendedparticulatematter (PM)(mg �m�3)were

also recorded. SO2 and PM concentrations in the urban air

were provided from continuous monitoring sites operated

by the Head Office of the H|fz|s|hha Institute (Public

Health Institute) in Eskisehir monthly (Table 2). Table 2

gives the monthly and annual mean of SO2 and PM con-

centrations in Eskisehir from March to November 2001.

Statistical Analysis

Statistical analysis (multivariate analysis (MANOVA))

of the data including fungal and actinomycetes colony

numbers and meteorological factors was performed.

Results

A total of 2518 fungal and 465 actinomycetes colonies

were isolated from 420 Petri dishes, quantified to deter-

mine the frequency of occurrence and then identified

(excepting the actinomycetes) (Tables 4 and 5). Some 20

fungal species could be identified, among them the species

Alternaria alternata that was generally found as the

predominant fungus (13.66%) at all sites, followed by

Cladosporium cladosporioides (5.80%) and Scopulariopsis

brevicaulis (5.50%) (Table 5). A. alternata, Aspergillus

wentii, C. cladosporioides, Penicillium chrysogenum,

S. brevicaulis and Ulocladium tuberculatum were found at

all sampling sites.

During the nine months of 2001 which were monitored,

the maximum concentration of airborne actinomycetes

was found in September (19.78%). The number of fungal

colonies on culture media plates was between 69 to a

maximum count of 553. The corresponding numbers

for actinomycetes were 20 and 92. In total 77.49% of the

fungal species could be identified in this study. The

remainder (22.51%) could not be identified using the

literature sources available because some of the genera

have similar morphological aspects, or no distinctive prop-

erties or there had been bacterial contamination (Table 5).

Statistical analysis of the data showed that the numbers

of fungi did not vary significantly by months and by areas

NOVEMBER

OCTOBER

SEPTEMBER

AUGUST

JULY

JUNE

MAY

APRIL

MARCH

Cou

nt o

f Act

inom

ycet

es (

cfu/

plat

e/15

min

)

10

8

6

4

2

0

Fig. 2. Actinomycetes concentrations according to the months(2001).

AU Iki E

ylul

AU Yun

usem

re C

.

OGU Mes

elik C

.

Cou

nt o

f Act

inom

ycet

es (

cfu/

plat

e/15

min

) 10

8

6

4

2

0

Fig. 3. Actinomycetes concentrations in research stations (1st, 2ndand 3rd stations, respectively).

Fungi and Actinomycetes in Eskisehir City Indoor Built Environ 2004;13:63–74 67

( p>0.05), while for the number of actinomycetes these

were significant ( p<0.001 for months and p<0.01 for

areas, respectively). For the numbers of actinomycetes,

March was different from the months of April, May, June,

July, August and November; April was different from

June, August, September, October and November; May

was different from June, August, September, October and

November; July was different from August, September

and October. Numbers of fungi differed between the 1st

and 2nd, and between the 2nd and 3rd areas (Figures 2 and

3). There was a negative correlation between the amount

of SO2 and the temperature (correlation coefficients are:

r¼�0.882, p¼ 0.002); there was a negative correlation

between the amount of PM and temperature (correlation

coefficients are: r¼�0.907, p¼ 0.001); there was a positive

correlation between the amount of SO2 and PM (correla-

tion coefficients are: r¼ 0.847, p¼ 0.004); there was a

negative correlation between the amount of SO2 and the

amount of sunshine (correlation coefficients are:

r¼�0.802, p¼ 0.009); there was a positive correlation

between the amount of PM and the relative humidity

(correlation coefficients are: r¼ 0.714, p¼ 0.031); finally,

there was a negative correlation between the amount of

sunshine and the amount of PM (correlation coefficients

are: r¼�0.844, p¼ 0.004).

High fungal spore densities were observed in

September, May and November (these were 21.96, 19.10

and 13.34%, respectively of the total CFU measured over

Table 3. Monthly meteorological parameters (Source: The Directorate of Eskisehir Meteorological Office)

Average monthlytemperature

(�C)

Total monthlyrainfall(mm)

Average monthlywind velocity(m � sec�1)

Average monthlyrelative humidity

(%RH)

Average monthlyhours of sunshine

(h)

Months 1941–2001

01.2001–12.2001

1941–2001

01.2001–12.2001

1985–2001

01.2001–12.2001

1933–2001

01.2001–12.2001

1985–2001

01.2001–12.2001

January �0.2 2.4 31.4 7.0 2.9 2.9 81.0 71.3 2.5 2.6February 1.2 2.7 27.7 14.6 3.2 3.4 77.0 65.0 4.0 4.2March 4.8 10.3 29.5 23.7 3.5 3.7 70.0 56.1 5.3 6.0April 10.2 10.3 30.7 66.8 3.3 3.6 64.0 62.3 6.1 6.4May 15.0 14.1 36.4 37.5 3.2 4.1 63.0 58.2 8.4 7.9June 18.7 20.2 28.5 0.0 3.3 3.9 59.0 40.7 10.1 11.1July 21.4 23.8 20.2 23.9 36. 4.4 54.0 48.8 11.2 11.3August 21.2 22.2 5.4 15.2 3.4 3.4 55.0 52.8 11.4 10.4September 17.1 18.3 11.5 6.3 2.9 2.9 60.0 53.3 9.0 9.4October 12.0 11.3 20.0 0.2 2.4 2.8 66.0 55.0 6.0 7.9November 6.7 5.6 24.4 95.7 2.7 3.1 74.0 68.7 4.2 3.0December 2.2 1.5 38.4 108.0 3.2 3.0 81.0 65.7 2.1 1.7Average 10.8 11.9 25.3 33.2 3.1 3.4 67.0 58.2 6.4 6.8

Total(for rainfall)

304.1 398.9

Table 2. Concentrations of SO2 and PM (aerosol) in the urban air in 2001 of Eskisehir city determined monthly (Source: Directorate ofEnvironment in Eskisehir province, Turkey)

Month Sep. Oct. Nov. Mar. Apr. May Jun. Jul. Aug. ANM#

SO2 (mg �m�3) 32 44 48 46 43 34 39 31 29 38.4

PM* 29 47 47 38 36 28 22 22 22 32.3

*PM: Particulate Matter in the air (¼ aerosol) (mg �m�3); # ANM: Average for the 9 months.

Permitted maximum levels for comparison (mg �m�3)

A. Short term (average of 24 h): SO2 400PM 300

B. Long term (annual average): SO2 150PM 150

C. Winter period (between October and March): SO2 400PM 300

68 Indoor Built Environ 2004;13:63–74 Asan et al.

the 9 month period); whereas the concentration of

actinomycetes was found to be maximum in September,

October and August (percentages were 19.78, 17.84 and

13.97%, respectively). The months in which least fungi

were observed were April, March and June (percentages

were 2.74, 5.04 and 5.79%, respectively); and for

actinomycetes in April, May and November (percentages

were 4.30, 4.30 and 6.88%, respectively).

Table 4. Distribution of the number of the samples taken and the average fungi according to months

Number of fungal colony Number of actinomycetes colony

(CFU/plate/15min) (CFU/plate/15min)

Months site 1 site 2 site 3 average Total % site 1 site 2 site 3 average Total %

March ’01 19 57 51 42.33 127 5.04 14 18 14 15.33 46 9.8924.3.2001 8 49 39 8 9 331.3.2001 11 8 12 6 9 11

April ’01 33 25 11 23.00 69 2.74 5 13 2 6.66 20 4.3008.4.2001 19 0 3 0 1 117.4.2001 10 9 5 5 7 122.4.2001 1 2 0 0 3 027.4.2001 3 14 3 0 2 0

May ’01 111 100 270 160.33 481 19.10 1 10 9 6.66 20 4.3005.5.2001 36 19 0 0 0 014.5.2001 66 32 217 0 0 021.5.2001 0 6 2 0 1 328.5.2001 9 43 51 1 9 6

June ’01 55 83 8 48.66 146 5.79 17 31 12 20.00 60 12.9004.6.2001 29 27 0 8 9 211.6.2001 19 43 2 7 12 217.6.2001 0 3 0 0 2 024.6.2001 7 10 6 2 8 8

July ’01 58 82 124 88.00 264 10.48 19 18 10 15.66 47 10.1001.7.2001 13 13 23 5 4 508.7.2001 20 55 22 3 7 015.7.2001 23 9 79 7 0 322.7.2001 2 5 0 4 7 229.7.2001 0 0 0 0 0 0

Aug. ’01 67 75 154 98.66 296 11.75 22 31 12 21.66 65 13.9704.8.2001 11 15 2 2 5 111.8.2001 6 0 1 8 9 518.8.2001 21 44 16 9 11 125.8.2001 29 16 135 3 6 5

Sept. ’01 119 394 40 184.33 553 21.96 19 36 37 30.66 92 19.7801.9.2001 10 22 0 6 12 1207.9.2001 62 26 9 6 6 014.9.2001 11 169 6 6 6 1221.9.2001 17 40 9 1 12 1228.9.2001 9 137 16 0 0 1

Oct. ’01 32 175 39 82.00 246 9.76 18 41 24 27.66 83 17.8405.10.2001 9 69 2 3 12 412.10.2001 0 37 14 2 9 617.10.2001 10 42 0 5 4 024.10.2001 13 27 23 8 16 1431.10.2001 0 0 0 0 0 0

Nov. ’01 69 108 159 112.00 336 13.34 6 14 12 10.66 32 6.8807.11.2001 70 96 133 6 11 914.11.2001 9 12 26 0 3 3

Total 563 1099 856 839.33 2518 – 121 212 132 155.00 465 –Percentage (%) 22.35 43.64 33.99 26.02 45.59 28.38

Fungi and Actinomycetes in Eskisehir City Indoor Built Environ 2004;13:63–74 69

Table 5. Colony numbers, percentage (%), sampling sites of fungal species and the months during which the samples were found (period:March–November 2001)

Genera and species Sites at which thesamples weretaken

Number ofcolonies

% ofTotal

Sampling months

Alternaria Nees 1,2,3 480 19.06A. alternata (Fr.) Keissl. 1,2,3 344 13.66 M (1-4, 2-6, 3-7), A (1-8, 2-1, 3-3), Y (1-10, 2-16,

3-10), U (1-7), L (1-18, 2-5, 3-25), G (1-5, 2-14,3-15), S (1-31, 2-57, 3-11), C (1-10, 2-34, 3-8),N (1-11, 2-18, 3-10).

A. citri Ellis & N. Pierce 1, 2, 3 60 2.38 M (2-3, 3-1), A (2-2), U (2-1), G (1-5, 2-7),S (1-2, 2-4), C (1-7, 2-5), N (1-5, 2-7, 3-11).

Aspergillus Fr.: Fr. 26 1.03A. niger Tiegh. 1, 2 11 0.43 Y (1-2, 2-1), U (1-1), L (2-1), G (1-1, 2-2), S (2-3)A. versicolor (Vuill.) Tirab. 2 1 0.04 G (2-1)A. wentii Wehmer 1, 2, 3 4 0.16 G (3-2), S (2-1), C (1-1)Chaetomium Kunze 2 0.08C. globosum Kunze 1 2 0.08 M (1-2)Cladosporium Link 218 8.66C. cladosporioides (Fresen.)G. A. de Vries

1, 2, 3 146 5.80 U (1-3), L (2-4), G (3-7), S (1-16, 2-15, 3-4),C (1-16, 2-21, 3-8), N (1-10, 2-8, 3-20).

Penicillium Link: Fr. 277 11.00P. chrysogenum Thom 1, 2, 3 54 2.14 A (1-2, 2-5, 3-3), M (3-5), G (2-5, 3-2), Y (1-10,

2-8, 3-11, L (3-3).P. citrinum Thom 2 4 0.16 Y (2-4)P. crustosum Thom 1, 2, 3 41 1.63 M (1-2), A (2-6), Y (1-6, 2-2, 3-25).P. decumbens Thom 1, 2 14 0.55 M (2-2), L (2-5), S (1-3), C (1-2), N (1-2).P. griseofulvum Dierckx 2, 3 3 0.12 M (2-1), Y (2-1), C (3-1).P. griseoroseum Dierckx 3 4 0.16 Y (3-1), M (3-3)P. puberulum Bainier 1, 2, 3 26 1.63 N (1-5, 2-2, 3-19).P. verrucosum Dierckx 3 5 0.20 Y (3-5)P. viridicatum Westling 2, 3 6 0.23 A (2-3), G (3-1), N (3-2).Scopulariopsis Bainier 163 6.47S. brevicaulis (Sacc.) Bainier 1, 2, 3 138 5.48 M (2-5), A (1-2), Y (2-9, 3-93), L (1-3, 2-6),

S (2-5), C (2-12, 3-3).Trichothecium Link 2 0.08T. roseum Pers. Link 1 2 0.08 C (1-2).Ulocladium Preuss 59 2.34U. tuberculatumE.G. Simmons

1, 2, 3 59 2.34 Y (1-7, 2-6), S (2-20), C (2-23, 3-3).

WardomycesF.T. Brooks & Hansf.

3 0.12

W. ovalis W. Gams 1 3 0.12 U (1-1), L (1-2).

Some species identified at the genus levelAlternaria sp. 1, 2, 3 76 3.02 Y (3-12), U (2-3, 1-2), L (3-5), G (1-3, 3-9), S

(2-1, 3-2), C (1-3, 2-6), S (1-7, 2-21), N (1-1, 2-1).Aspergillus sp. 1, 2, 3 10 0.40 N (1-3, 2-2, 3-5).BasipetosporaG.T. Cole & W.B. Kendr.

1, 2, 3 23 0.91 M (3-11), L (1-5), C (2-7).

Cladosporium sp. 1 72 2.86 M (1-2, 3-2), Y (1-7, 2-8, 3-15), U (1-3), G (1-3,3-8), S (1-5), C (1-3, 2-3), N (1-3, 3-10).

Fusarium Link sp. 1 2 10 0.40 M (2-3), G (2-3), S (2-2), N (2-2).Fusarium sp. 2 2 17 0.67 M (2-2), G (3-5), S (1-2, 2-5), N (2-3).Mycelia sterilia 1, 2, 3 649 25.77 M (1-4, 2-16, 3-10), A (1-9, 2-7), Y (1-13, 2-32,

3-52), U (1-19, 2-42, 3-5), L (1-13, 2-28, 3-36),G(1-13, 2-20, 3-40), S (1-31, 2-119, 3-13), C (1-14,2-38, 3-9), N 1-20, 2-20, 3-26).

Penicillium sp. 1 1, 2, 3 18 0.71 M (3-3), Y (1-6, 3-7), U (2-2).Penicillium sp. 2 1, 2, 3 82 3.26 M (2-5), A (1-2, 3-1), Y (1-22, 2-5, 3-12), U (1-3),

L (1-6, 2-3), G (3-11), S (3-1), C (2-3), N (3-8).

(Continued )

70 Indoor Built Environ 2004;13:63–74 Asan et al.

Discussion

The present study will contribute to our knowledge of

the levels and types of airborne fungi and actinomycetes in

the urban air of Eskisehir. Although similar studies [22, 23]

have been undertaken in this city, one of these [23] focused

on airborne bacteria and the other [22] looked at airborne

fungi, some of which were determined only to genus level.

New studies are needed because of the rapid development

in urbanisation, with concomitant increasing air pollution

and industrialisation. One of the studies noted above [22]

was published nine years ago and it contains no data

related to concentration and distribution of airborne

fungal species. In the present study, concentrations were

measured and several of the fungal species were identified.

Unfortunately, the nature of mycoflora is such that it was

difficult to specify all the airborne fungi found.

It is well known that microfungi can live under extreme

conditions in almost all regions and all climates. They

usually live in soil and can be dispersed into the atmos-

phere under the influence of various factors. In recent

years, aerobiologists have shown a great interest in

airborne fungi due to both their ubiquitous presence in

the air and the increase in allergies attributed to them

[9,44]. Monitoring airborne fungi in both outdoor and

indoor environments provides valuable data for evaluating

type and distribution. Aspergillus and Penicillium spores

are the most widespread aeroallergens in the world.

According to qualitative and quantitative reports, the

first is the dominant species in tropical regions while the

latter is dominant over the rest of the world [45].

Everybody may be exposed to moulds, but fungal density

in the air and the time for which people are exposed are

both important for assessing any adverse effect of moulds.

In this study Alternaria was the most frequent and

the predominant genus detected, followed by Penicillium,

Cladosporium and Scopulariopsis genera (Table 5).

According to Gambale et al. [46], the genus Alternaria

with powerful allergenic properties has been isolated with

16% frequency in sampled collections. Downs et al. [47]

noted that Alternaria is known to be allergenic and is

one of the most common fungi world-wide and they have

suggested that Alternaria allergens contribute to severe

asthma. Myszkowska et al. [48] noted that some 4–7% of

the European population shows sensitivity to Alternaria

and Cladosporium spores. C. cladosporioides, determined

in our study, has been reported to be the agent

causing phaeohyphomycosis along with other species of

Cladosporium [49]. Also we identified Aspergillus niger

which is well-known to cause many health problems

such as extrinsic alveolitis, allergic bronchopulmonary

aspergillosis, keratitis, endophthalmitis, primary cuta-

neous aspergillosis and necrosing otitis in humans [50].

Although the prevalence of fungal sensitivity in asthma

is not completely understood, A. alternata species

(which were found as an appreciable fraction of the

whole in our study) are a common cause of asthma [51].

Although spore numbers of Alternaria sp. lower than those

of Cladosporium sp. have been found in some studies

[15,52], the spore volume of Alternaria sp. is greater than

Cladosporium sp., so together they are comparable in

biomass (Dr. Julie M. Corden, Derby-UK, personal

communication). Alternaria, Penicillium, Aspergillus and

Fusarium genera were found to be the dominant fungi in

some studies such as the one by Savino and Caretta [53].

Cladosporium sp. is the most common fungus living as a

saprophyte, mainly on dying and/or dead herbaceous

plants and other organic matter. It produces chains of dry

Table 5. Continued

Genera andspecies

Sites at which thesamples weretaken

Number ofcolonies

% ofTotal

Sampling months

Penicillium sp. 3 1, 2, 3 20 0.79 M (3-3), A (3-2), G (2-8), Y (1-2), S (2-2), N (2-3).Scopulariopsis sp. 2, 3 25 0.99 A (3-2), Y (2-1), S (2-15), C (2-4, 3-3).Torula Pers. sp. 1, 2, 3 22 0.87 A (1-3), M (1-4), G (2-2, 3-1), S (3-4), C (1-5),

N (1-3).Unidentified 1, 2, 3 567 22.51 M (1-3, 2-15, 3-9), A (1-7, 2-2, 3-3), Y (1-10,

2-23, 3-42), U (1-8, 2-23,3-3), L (1-8, 2-30, 3-41),G (1-17, 2-13, 3-53), S (1-12, 2-109, 3-7),C (1-14, 2-23, 3-6), N (1-12, 2-30, 3-44).

Total 2518

Letters indicate: M: March, A: April, Y: May, U: June, L: July, G: August, S: September, C: October, N: November. The first

(and second and third where appropriate) number in parenthesis in the last column is the station number with related month.

Fungi and Actinomycetes in Eskisehir City Indoor Built Environ 2004;13:63–74 71

conidia that easily become airborne (Dr. Hugues Beguin,

Brussels – Belgium; personal communication).

Although there are different methods for sampling

fungi from air, we used the Petri Plate Gravitational

Settling Method for the isolation of airborne fungi and

actinomycetes because of its practicality and low cost. This

method is useful for the enumeration of fungal spores, but

gives only a rough approximation of the kinds and

numbers of airborne fungi [54]. According to Chen et al.

[55], there is no official and universally accepted bioaero-

sols sampling method. There are many types of sampler

(Rotorod, Tauber, Burjerd 7 day, personal Burkard, air-o-

cell, Andersen, SAS, etc.) for sampling aeroallergens from

air; but although each has its advantages and disadvan-

tages they all have some limitations [56]. In addition,

although many types of culture media are used for

sampling micro-organisms from air we used Rose-Bengal

streptomycin agar medium. According to Madan et al.

[57], this medium is the most suitable for sampling fungi

from air. Also according to Morring et al. [58], Rose-

Bengal streptomycin agar can be used for aeromycological

sampling. Streptomycin antibiotic was used to control

reproduction of bacteria and Rose-Bengal stain was used

to limit the growth of fast-growing moulds (e.g., Rhizopus

and Trichoderma spp.). Also, Wu et al. [59] reported that

dichloron (present in dichloron glycerol-18 agar¼DG18)

restricts the growth of fast-growing genera but we did not

use it. Wu et al. [60] compared some media for sampling

fungi from a hospital environment. They proposed DG18

medium for sampling but this medium has a low water

activity and is best used for xerophilic moulds and

osmophilic yeasts. Also Levetin and Horner [56] noted

that MEA is generally suggested for mesophilic fungi.

Sampling time was between 10.00 and 12.00 in our study

after Levetin and Horner [56] who pointed out that the

spores of many asexual fungi peaked in air in early to mid-

afternoon but were low in early morning.

Alternaria and Penicillium genera were found more

abundantly than other fungi at all the research stations.

A. alternata was present throughout the year but was at a

maximum in September, October and November. A. citri

occurred throughout seven months: it was not seen in

December and July but was especially abundant in

November. Corden and Millington [61] determined that

the Alternaria sp. spores peak daily count usually occurred

in August but occasionally in late July or early September.

Annual variations in the composition of the vegetation

around the research station may cause the differences.

Mitakakis and Guest [62] noted the seasonality of spore

levels of Cladosporium and Alternaria sp. which peaked in

spring and summer. Bandyopadhyay et al. [63] found a

positive correlation between heavy rainfall and Fusarium

sp. concentration. Other researchers [8,63] including Di

Giorgio et al. [10] reported that various meteorological

factors affected the type and concentrations of airborne

fungi. Among these wind velocity, relative humidity and

temperature were particularly important. Pasanen et al.

[64] reported that the minimum air velocity at which

Cladosporium sp. released spores was 1.0m � s�1, however

Aspergillus fumigatus and Penicillium sp. released great

numbers of spores at 0.5m � s�1. So, air velocity is

significant for dispersion of fungal spores into the air.

Reponen et al. [65] reported that the hygroscopicity of

fungal airborne spores significantly affected their aero-

dynamic diameter.

Aspergillus niger, A. versicolor, A. wentii and Penicillium

chrysogenum identified in our study are widespread in

Turkey and have been determined in other studies [66].

Some species of Cladosporium, Alternaria, Penicillium,

Fusarium and Aspergillus genera (determined in our study)

can cause allergy [67–69] and these genera are known to be

an important part of the airborne microfungi. Although

Aspergillus fumigatus is the most common agent of

invasive aspergillosis, A. niger, found in our study, has

also been implicated [70]. The airborne fungal species

identified in our study may be allergenic to people residing

in Eskisehir and the population may be exposed to dust

containing mycotoxins derived from fungi. Therefore,

fungal spore monitoring in Eskisehir city may be useful

from the allergological point of view. Future investigations

are needed to examine further the effects of mould

exposures on the related health problems.

We showed above that our study revealed the existence

of a rich population of airborne fungi and actinomycetes

in the urban air. However, it must be borne in mind that

the culture methods used reveal only a portion of the

airborne micro-organisms because some fungi are not able

to grow at all in culture media and some of them may lose

viability [56]. Therefore, the air of Eskisehir city may be

even richer in fungi and actinomycetes than our results

have shown.

72 Indoor Built Environ 2004;13:63–74 Asan et al.

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