ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 1355 1365

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Microorganisms in bioaerosol emissions from wastewater

treatment plants during summer at a Mediterranean site

Styliani Karra, Eleftheria Katsivela

Laboratory of Environmental Chemistry and Biochemical Processes, Department of Natural Resources and Environment, Technological
Educational Institute of Crete, Romanou 3, Halepa, GR 73133 Chania, Greece

art i cle info ab st rac t

Article history: Measurements were conducted at a Mediterranean site (latitude 351 310 north and longitude
Received 11 May 2006 241 030 east) during summer, to study the concentration of microorganisms emitted from a
Received in revised form wastewater treatment plant under intensive solar radiation (520840 W/m2) and at elevated
6 December 2006 air temperatures (2531 1C). Air samples were taken with the Air Sampler MAS 100 (Merck)
Accepted 7 December 2006 at each stage of an activated-sludge wastewater treatment (pretreatment, primary settling
Available online 2 February 2007 tanks, aeration tanks, secondary settling tanks, chlorination, and sludge processors).
Keywords: Cultivation methods based on the viable counts of mesophilic heterotrophic bacteria, as
Bioaerosols well as of indicator microorganisms of faecal contamination (total and faecal coliforms and
Wastewater enterococci), and fungi were performed. During air sampling, temperature, solar radiation,
Airborne bacteria relative humidity and wind speed were measured. The highest concentrations of airborne
Coliforms microorganisms were observed at the aerated grit removal of wastewater at the
Enterococci pretreatment stage. A gradual decrease of bioaerosol emissions was observed during the
Airborne fungi advanced wastewater treatment from the pretreatment to the primary, secondary and
tertiary treatment (97.4% decrease of mesophilic heterotrophic bacteria, and 100% decrease
of total coliforms, faecal coliforms and enterococci), 95.8% decrease of fungi. The
concentration of the airborne microorganisms at the secondary and tertiary treatment of
the wastewater was lower than in the outdoor control. At the same time, the reduction of
the microbial load at the waste sludge processors was 19.7% for the mesophilic
heterotrophic bacteria, 99.4% for the total coliforms, and 100% for the faecal coliforms
and the enterococci, 84.2% for the fungi. The current study concludes that the intensive
solar radiation, together with high ambient temperatures, as well as optimal wastewater
treatment are the most important factors for low numbers of microbes in the air.
& 2007 Elsevier Ltd. All rights reserved.

1. Introduction for the dissemination of human and animal pathogens (Pillai

Aerosolization of microbial pathogens, endotoxins, and
odours is an inevitable consequence of the generation and
handling of wastewater. High amounts of microorganisms are
not only present in the wastewater but also in bioaerosols
that are generated during the different phases of the process
in wastewater treatment plants. Bioaerosols can be a vehicle
and Ricke, 2002). Many studies have been carried out to
evaluate the biological risks of aerosols by determining the
concentrations of viable microorganisms, using different
sampling and detection methods (Crawford and Jones, 1979;
Buttner and Stetzenbach, 1991; Ranalli et al., 2000; Pillai and
Ricke, 2002; Pascual et al., 2003). However, significant knowl-
edge and technology gaps still exist. These include, among

Corresponding author. Tel.: +30 2821 0 23071; fax: +30 2821 0 23003.
E-mail address: katsivela@chania.teicrete.gr (E. Katsivela).
0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2006.12.014
 ARTICLE IN PRESS
1356 WAT E R R E S E A R C H
other things, the lack of a clear understanding for accurately
predicting the health risks associated with bioaerolized
pathogens. In order to ensure the health of workers and the
public, it is important to determine the composition and
concentration of airborne microorganisms in contaminated
environments. Moreover, the typical route of exposure to
organisms that are primarily associated with intestinal
infections, such as Salmonella spp. and enteric viruses, is
thought to be through inhalation in which pathogens are
deposited in the throat and upper airway and swallowed
(Wathes et al., 1988).
The transient concentration of outdoor atmospheric bac-
teria is the result of a dynamic equilibrium between the
bacterial input and output fluxes to and from the atmosphere
(Lighthart and Schaffer, 1994). In a wastewater treatment
plant, in summer, the input flux to the atmosphere is
primarily from the wastewater, while the output flux from
the air depends primarily upon damage and decay of the
microbes, transport through the wind, gravitational settling
(Gregory, 1973), dilution effects due to atmospheric mixed
layer events and adsorption on surfaces. Many parameters
are described as being potentially damaging and lethal for
microbial cells in the air. Some of these include solar
radiation (Maier et al., 2000), de- and re-hydration of bacterial
cells (Crowe et al., 1990; Israeli et al., 1993), extreme
temperature effects and certain physical and meteorological
factors, such as formation of radicals and ions, air turbulence
(Maier et al., 2000), gravitational settling (Gregory, 1973) and
dilution effects, due to atmospheric mixed layer events. On
the other hand, some of the above parameters contribute also
to the output of microbes from the wastewater to the
atmosphere. The presence of agitation devices at the waste-
water treatment plant, the temperature, the humidity, the air
turbulence and the wind speed are some of these parameters.
The aim of the present work was to determine the
concentrations of airborne microorganisms, which are in-
dicators for microbial load, in general, and particularly for
faecal contamination, in bioaerosols originating from muni-
cipal wastewater treatment plants at the different stages of
treatment. Especially, the interest was focused at a Mediter-
ranean site in the summertime (from May to July 2005), due to
the expected high evaporation of the wastewater surface and
the possible implicit high emission of bioaerosols.
2. Material and methods
2.1. Sampling
Bioaerosol samples were collected from May to July 2005 at
the Municipal Wastewater Treatment Plant of Chania (Crete,
Greece) with activated-sludge biological treatment. The
wastewater treatment plant, with a daily mean inflow of
17,150 m3/d, includes indoor (lodged in an extra building)
pretreatment with screens and coarse bubble aerated grit
chambers, outdoor partially covered primary settling tanks,
outdoor aeration tanks with fine bubble-diffusers, outdoor
secondary settling tanks, outdoor chlorination and indoor
sludge processors with belt filter presses.
41 (2007) 1355 1365
Air samples were collected by impaction with an air
sampler MAS 100 (Merck, Germany) at each wastewater
treatment stage. Impaction is the forced deposition of
airborne particles on a solid surface (Maier et al., 2000). The
air sampler MAS 100 was a single-stage impactor that
aspirated air through a 400-hole perforated plate. The
resulting air-stream, which contained particles with a dia-
meter equal to or larger than 1 mm, was directed onto 90 mm
diameter agar Petri dishes (Merck, 2002). The impaction flow
rate was 100 L/min and coincided with the level 5 of the
Andersen sampler. After every collection cycle, the Petri dish
was incubated and the colonies were counted and expressed
as colony-forming units per cubic metre (CFU/m3).
Duplicates of samples of mesophilic heterotrophic bacteria,
of indicator microorganisms of faecal contamination (total
coliforms, faecal coliforms and enterococci), and of fungi
were collected at each sampling time (12 campaigns in total)
at the following sampling sites of the Wastewater Treatment
Plant: screens, coarse bubble aerated grit chambers, primary
settling tanks, fine bubble aeration tanks, secondary settling
tanks, chlorination tanks, and sludge processors. In total, 24
samples of every measured microorganism category were
collected at each sampling site of the Wastewater Treatment
Plant during this study.
Measurements of the previously mentioned bacteria and
fungi at the indoor wastewater treatment stages as well as at
the outdoor treatment facilities were compared with an
indoor and an outdoor control, respectively. Thus, control
measurements were performed at a clean outdoor environ-
ment (a courtyard with small garden), located 5 km from the
measurement site, and at a busy, indoor area (a restaurant),
filled with people.
The volume of the collected samples was dependant on the
sampling site, the expected microorganism concentration
and the associated microorganisms. Thus, the collected air
volume varied from 50 to 2000 L. The exact volumes collected
are shown in Table 1.
During air sampling, temperature, relative humidity, wind
direction and speed, and solar radiation were measured. The
temperature (expressed in 1C) and the relative humidity
(expressed in %) were measured with a portable instrument
(Preservation Equipment Ltd, UK). The wind direction and the
wind speed (expressed in m/s) were determined by a portable
anemometer (Campbell Scientific, Inc., USA). The solar
radiation was measured hourly by a pyranometer (Kipp &
Zonen, Netherlands) and was expressed in W/m2. The dosage
of solar radiation was calculated as follows: dosage of solar
radiation (W min/m2) intensity (W/m2)  exposure time
(min). The dosage of solar radiation over a certain time
interval was calculated by adding the mean hourly dosages.
A mean hourly dosage was calculated by multiplying the
mean solar radiation during the hour by 60 min.
Wastewater samples from the different stages of treatment
were examined by the membrane filter technique, using
sterile membrane filters of 47 mm diameter with 0.45 mm pore
size, as described in the Standard Methods for Examination of
Water and Wastewater by the American Public Health
Association, the American Water Works Association and the
Water Environment Federation (1998). In this case, the results
were expressed in CFU per millilitre of wastewater.
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 135 5 136 5 1357

Table 1 Optimum impaction volume for each collected microorganism depending on the sampling site

Sampling site Heterotrophic bacteria (L) Total coliforms (L) Faecal coliforms (L) Enterococci (L) Fungi (L)

Screens 250 500 2000 2000 250

Aerated grit chambers 50 250 2000 2000 50
Primary settling tanks 500 2000 2000 2000 500
Aeration tanks 1000 2000 2000 2000 1000
Secondary settling tanks 1000 2000 2000 2000 1000
Chlorination 1000 2000 2000 2000 1000
Outdoor control 100 2000 2000 2000 100
Sludge processors 50 2000 2000 2000 50
Indoor control 50 2000 2000 2000 50

The daily wastewater inflow (expressed in m3/d) was
measured by an ultrasonic flow measurement instrument
(Endress and Hauser, Germany) at the wastewater treatment
plant.
2.2. Microbial enrichment and cultivation
parameters for the calculation of the predicted microorgan-
ism concentrations. Wind speed of 0 m/s was used for the
calculation of the log (CFU/m3) in the pretreatment and in the
sludge processors because these facilities were indoors,
lodged in extra buildings.

Airborne microorganisms were enriched from the collected 3. Results

air samples in 90 mm Petri dishes containing different agar
media and were cultivated as follows. The mesophilic
heterotrophic bacteria were incubated in Nutrient Agar
(Merck, Germany) at 37 1C for 48 h. The total and faecal
coliforms were cultivated in Membrane Lauryl Sulphate Broth
(Lab M, England) supplemented with 1.5% agar (Fluka,
Germany) for 24 h at 37 1C and at 44 1C, respectively. Only
yellow-coloured colonies were counted as coliforms in both
cases. The enterococci were incubated in Slanetz and Bartley
Medium (Lab M, England) at 37 1C for 48 h. All red and maroon
colonies were counted as presumptive enterococci. Fungi
were cultivated in Malt Extract Agar (Lab M, England) at 25 1C
for 72 h. Reliable results were obtained only when the colony
number per plate (90 mm) did not exceed the number 80.
The above-mentioned culture media were also used for the
determination of all measured bacteria and fungi in the
wastewater samples.
2.3. Mathematical model used
The concentrations of the counted heterotrophic bacteria, the
fungi and the indicators for faecal contamination (total and
faecal coliforms) in the air samples from different stages of
the wastewater treatment plant were compared with a
mathematical model for the prediction of microorganism
concentrations in bioaerosols (Pascual et al., 2003). For the
application of this linear model, the concentrations of the
heterotrophic bacteria, the fungi and the total and faecal
coliforms at the following four different locations of the
wastewater treatment plant were used: aerated grit chambers
(pretreatment), primary settling tanks (primary treatment),
aeration tanks (secondary treatment) and belt filter presses
(sludge processors). Besides the concentrations of microor-
ganisms in the bioaerosol emissions at the above locations,
the daily inflow at the wastewater treatment plant and the
wind speed during sampling were also required as numerical
3.1. Determination of airborne microorganisms
At the beginning of this study, the optimization of the proper
sampling volume for impaction was necessary for the
determination of specific components of the bioaerosols
emitted during the biological wastewater treatment. A colony
overlapping, due to high impaction volumes, as well as no
detection of airborne microorganisms, due to low impaction
volumes, was considered and these measurements were
avoided for reliable results by the culture-based detection.
Optimum measurements at the different stages of the
wastewater treatment were obtained using impaction vo-
lumes in the range of a minimum of 50 L by the heterotrophic
bacteria and fungi at the grit removal and sludge processor
facilities to a maximum of 2000 L for the indicator micro-
organisms for faecal contamination at the secondary and
tertiary wastewater treatment (Table 1).
The results reported for each microorganism category in
every sampling site are the average (arithmetic mean
concentration) and standard deviation of the counts obtained
from the 12 morning sampling campaigns. The final results
were expressed in CFU/m3. The highest concentrations of
microbial load were detected at the stage of pretreatment
of the wastewater (at the screens and, especially, at the
aerated grit removal; Fig. 1 and Table 2). The measured
mean concentrations at the aerated grit removal were as
follows: mean concentration of mesophilic heterotrophic
bacteria were 9337636 CFU/m3; mean concentration of total
coliforms 1727107 CFU/m3; mean concentration of faecal
coliforms 28734 CFU/m3; mean concentration of enterococci
56717 CFU/m3; mean concentration of fungi 3807200 CFU/
m3. In comparison, the mean concentrations of the hetero-
trophic bacteria and fungi at the indoor control (heterotrophic
bacteria: 5157295 CFU/m3; fungi: 160750 CFU/m3; Fig. 1)
were approximately 50% lower than at the aerated grit
 ARTICLE IN PRESS
1358 WAT E R R E S E A R C H 41 (2007) 1355 1365

A
Indoor control

Sludge processors

Outdoor control
Sampling sites

Chlorination tanks

Secondary settlers

Aeration tanks

Primary settlers

Grit removal

Screens

0 500 1000 1500 2000 2500

Mesophilic heterotrophic bacteria (cfu/m3)

B
Indoor control

Sludge processors

Outdoor control
Sampling sites

Chlorination tanks

Secondary settlers

Aeration tanks

Primary settlers

Grit removal

Screens

0 100 200 300 400 500 600 700

Fungi (cfu/m3)

Fig. 1 Mean concentrations of (A) mesophilic heterotrophic bacteria and (B) fungi in bioaerosols at different stages of a
wastewater treatment plant. Error bars indicate minimum and maximum counts.

Table 2 Arithmetic means, minimum and maximum concentrations of indicators of faecal contamination in bioaerosols
at different stages of a wastewater treatment plant

Sampling site Total coliforms (CFU/m3) Faecal coliforms (CFU/m3) Enterococci (CFU/m3)

Mean Min Max Mean Min Max Mean Min Max

Screens 10 1 17 8 0 12 9 0 14
Aerated grit chambers 172 32 340 28 1 93 45 23 65
Primary settling tanks 16 1 76 2 0 6 3 0 5
Aeration tanks 1 0 1 0 0 0 0 0 0
Secondary settling tanks 1 0 5 0 0 0 0 0 0
Chlorination 0 0 0 0 0 0 0 0 0
Outdoor control 2 0 9 0 0 0 0 0 0
Sludge processors 1 0 3 0 0 0 0 0 0
Indoor control 4 0 14 0 0 0 2 0 5

Error bars indicate minimum and maximum counts.

 ARTICLE IN PRESS
WAT E R R E S E A R C H
removal. At the same time, the mean concentrations of the
indicator microorganisms of faecal contamination at the
indoor control (total coliforms: 476 CFU/m3; faecal coliforms:
070 CFU/m3; enterococci: 272 CFU/m3; Table 2) were at least
96% lower than at the aerated grit removal. In comparison, the
concentrations of the heterotrophic bacteria, the total coli-
forms, the faecal coliforms, the enterococci, and the fungi in
the wastewater sampled from the aerated grit removal were
7.35  106 CFU/mL (7.35  1012 CFU/m3), 1.6  104 CFU/mL (1.6 
1010 CFU/m3), 2  103 CFU/mL (2  109 CFU/m3), 2.5  104 CFU/mL
(2.5  1010 CFU/m3), and 7 CFU/mL (7  106 CFU/m3), respec-
tively. Thus, the concentration of the measured bacteria in
the wastewater was, at least, 108 times higher than the
concentration in the air. In contrast, the concentration of the
measured fungi in the wastewater was 106 times lower than
the concentration in the air.
A gradual decrease of the concentration of the hetero-
trophic bacteria as well as of the fungi in the bioaerosols was
obtained by advanced treatment of wastewater (secondary
and tertiary treatment) (Fig. 1). The concentration of the
heterotrophic bacteria and the fungi decreased more than
50% after the pretreatment of the wastewater. The concen-
tration of the airborne microorganisms in the outdoor control
was even higher than those at the secondary and tertiary
treatments of the wastewater (Fig. 1). In total, 97.4% decrease
of the heterotrophic bacteria and 95.8% decrease of the fungi
was achieved.
Comparable results were obtained also with respect to the
indicator microorganisms of faecal contamination (Table 2).
In this case, the pathogens tended towards zero in the
bioaerosols at the aeration tanks and were not more
detectable at the secondary settlers or at the disinfection
tanks. A mean concentration of 1 CFU/m3 of total coliforms,
that was often as high as in the clean outdoor control, was
detectable at the aeration tanks. Faecal coliforms and
enterococci could never be detected in air samples of 2000 L
at the secondary settling tanks or at the disinfection tanks
(Table 2). In comparison, concentrations of 2.4  103 CFU/mL
(2.4  109 CFU/m3), 7  102 CFU/mL (7  108 CFU/m3)and 6.5 
103 CFU/mL (6.5  109 CFU/m3) for the total and faecal coli-
forms and the enterococci, respectively, were determined in
the secondary treated wastewater before disinfection. Thus, a
total of 100% decrease for the indicator microorganisms of
faecal contamination was achieved.
Concentrations of airborne microorganims were also de-
termined in the air inside the building with the belt filter
presses of the waste sludge processors. As shown in Fig. 1, the
mean concentration of the airborne heterotrophic bacteria
was lower than their concentrations at the pretreatment and
primary settlers but higher than at all residual treatment
stages of the wastewater. However, the concentrations of
heterotrophic bacteria at the sludge processors were lower
than the concentrations of a busy indoor place used as
control. In contrast, the counts of the airborne fungi at
the sludge processors were the second highest after their
counts at the grit removal and remarkably higher than
the concentrations at the indoor control. Regarding the
indicator microorganisms, only low concentrations of total
coliforms (1 CFU/m3) were measured. Faecal coliforms and
enterococci could not be detected in air samples of 2000 L. So,
4 1 (200 7) 135 5 136 5 1359
the decrease of bioaerosol emission from the pretreatment to
the sludge processors was 19.7% for the heterotrophic
bacteria, 99.4% for the total coliforms, 100% for the faecal
coliforms and the enterococci and, finally, 84.2% for the fungi
(Fig. 1 and Table 2).
3.2. Influence of meteorological conditions
As mentioned before, sampling of bioaerosols was carried out
during summer 2005 at a location with latitude 351 310 north
and longitude 241 030 east, where the solar radiation was
intense and the air temperature was high. Fig. 2 shows the
mean hourly meteorological conditions of temperature and
solar radiation during the observation periods. The sampling
time was usually from 10 a.m. to 3 p.m. when the solar
radiation increased roughly from 520 to 840 W/m2. The
ambient temperature in the outdoor facilities ranged
from 25 to 31 1C and the relative humidity at approximately
40%. In order to determine the influence of the solar
radiation, the temperature and the relative humidity to
microorganism concentrations in the air, two sampling
campaigns were performed in two different days in the
morning/noon (from 10 to 3 p.m.) and in the afternoon/
evening (from 4 to 9 p.m.). During the afternoon/evening
sampling campaigns the solar radiation decreased from
730 to 10 W/m2, whereas the ambient temperature ranged
from 29 to 27 1C and the relative humidity at approximately
55%. Duplicate samples of bacteria (heterotrophic bacteria,
total coliforms, faecal coliforms and enterococci), as well
as of fungi, were collected at each sampling time. The
changes of the mean concentrations of airborne microorgan-
isms from the two compared morning and two afternoon
campaigns, according to the changes in air temperature,
solar radiation and relative humidity at outdoor facilities of
the wastewater treatment plant are summarized in Fig. 3.
Thereby, an increase in the airborne bacterial concentrations
between 2 and 9 times was observed at the outdoor facilities
when the ambient temperature and the solar radiation
decreased. In contrast, a decrease in the airborne fungal
concentrations at the wastewater treatment facilities
was observed. The fungal concentrations were from 2 to 9
times lower in the afternoon, in comparison to the concen-
trations at noontime, with the highest solar radiation. In
comparison to the data of the outdoor facilities, the concen-
trations of the airborne microorganisms at the indoor
facilities were within the expected range.
The effect of the changes in solar radiation, air temperature
and relative humidity on the bacterial and fungal counts in
the air was assessed by additional measurements at the
outdoor control. Thereby, the outdoor concentrations of
airborne microorganisms were monitored at morning, noon
and evening during five different summer days (Fig. 4).
Measurements of airborne heterotrophic bacteria and fungi
in the outdoor clean control area at the same time period
attempted to detect the microbial flux in the air under similar
environmental conditions. As shown, a decrease in the
airborne heterotrophic bacteria concentration from morning
to noon was accompanied by an increasing dosage of
solar radiation (at sunshine: 41,640 W  min/m2 at 10 a.m.;
170,562 W  min/m2 at 1 p.m.; 352,195 W  min/m2 at 6 p.m.).

1360
900
800
700
Solar radiation (W/m2)
600
500
400
300
200
100
0 0
5:00 6:00 7:00 8:00
Fig. 2 Mean hourly meteorological conditions of temperature and solar radiation during the observation periods.
Thus, during sunshine, a decrease of bacterial concentrations
of approximately 7.8 times was achieved between 10 and
1 p.m. Thereafter, a slower decrease in bacterial concentra-
tions of approximately 2.4 times between 1 and 6 p.m. with
decreasing solar radiation and temperature, together
with increasing relative humidity, was observed. In contrast,
the airborne fungal concentrations showed a maximum at
noon time (1 p.m.). Thus during sunshine, an increase of
fungal concentrations of approximately 1.6 times was
achieved between 10 and 1 p.m., by increasing the dose of
solar radiation. Finally, with a further increase in the dosage
of solar radiation, a decrease in fungal concentrations of
approximately 1.6 times was obtained, bringing the fungal
concentrations to the morning values (Fig. 4).
3.3. Model comparison
As mentioned in Section 2.3, a linear model for predicting
concentrations of microorganisms in bioaerosol emissions
at wastewater treatment plants (Pascual et al., 2003) was used
to correlate the measured and the calculated concentrations
of the airborne microorganisms. However, a low correlation
between the calculated data and the measured concentra-
tions of airborne bacteria during summer at the Mediterra-
nean site under study at different stages of the wastewater
treatment plant was obtained (Fig. 5). The measured data,
from morning and afternoon samplings, which were taken at
different days, differed from the calculated by 12 orders of
magnitude. Additionally, the measured data could not con-
firm the prediction of the model, that with increasing inflow
of wastewater, a higher concentration of bioaerosol should
be expected. As shown in Fig. 5, the daily inflow of waste-
water from the presented morning sampling was higher
(18,065 m3/d) than the inflow from the afternoon sampling
(16,832 m3/d). The high calculated microbial concentrations
during morning sampling were correlated with elevated
wastewater inflow (Fig. 5). However, a gradual decrease of
ARTICLE IN PRESS
WAT E R R E S E A R C H 41 (2007) 1355 1365
35
30
25
Temperature (C)
20
15
10
Solar radiation (W/m2)
Temperature (C)
5
9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00 20:00 21:00
Time of day
airborne microbes with advanced wastewater treatment, as
well as an increase between the concentrations at the
secondary wastewater treatment and at the sludge proces-
sors, was confirmed in the calculated as well as in the
measured data (Fig. 5). Regarding the concentrations of the
total and faecal coliforms and enterococci (data not shown),
the slight increasing tendency in the calculated concentra-
tions between the secondary wastewater treatment and the
sludge processors could not be supported by the measured
data, probably, due to the low detection of pathogens at these
two stages (Table 2).
As shown in Fig. 5, the agreement of the measured with the
calculated data regarding the tendency was better during the
afternoon sampling than during the morning sampling.
Differences in the meteorological conditions (solar radiation,
temperature and relative humidity) may be responsible
for this.
4. Discussion
The air sampler MAS 100 is made to comply with ISO/CD
14698-1:1996 recommendations for clean rooms and asso-
ciated controlled environments (Merck, 2002). A number of
cultivation-based approaches are available to detect and
characterize the viable components of bioaerosols (Marthi
et al., 1990; Stewart et al., 1995; Ranalli et al., 2000). In the
present study, cultivation-based detection in complete and
selective media directly after impaction was used for the
determination of specific components of bioaerosols emitted
during the biological wastewater treatment. Mesophilic
heterotrophic bacteria were chosen to be analysed because
of their prevalence in the environment, including among
others bacterial genera such as Pseudomonas, Aeromonas,
Klebsiella, Bacillus, Staphylococcus, Streptococcus, Escherichia,
Enterobacter, Proteus, Alcaligenes, as well as Stenotrophomonas.
Some members of these genera are opportunistic pathogens
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 135 5 136 5 1361

Morning sampling
900 900

800 800

700 700

Solar Radiation (W/m2)

Concentration (CFU/m3)
Heterotrophic Bacteria
Fungi
600 Total Coliforms 600
Enterococci
Faecal coliforms
500 Solar Radiation 500

400 400

300 300

200 200

100 100

0 0
s

l
ro
er

nk

er

nk

nt
ttl

ttl
Ta

Ta

Co
Se

Se
n

n
io

io
y

ry

or
ar

at

at
da

do
im

er

in
n

ut
A

r
co
Pr

lo

O
Se

900
Afternoon sampling Ch 900

800 Heterotrophic Bacteria 800

Fungi
Total coliforms
700 Enterococci 700
Concentration (CFU/m3)

Faecal coliforms

Solar Radiation (W/m2)

Solar Radiation
600 600

500 500

400 400

300 300

200 200

100 100

0 0
s

l
ro
er

nk

er

nk

nt
ttl

ttl
Ta

Ta

Co
Se

Se
n

n
io

io
y

or
ar

ar
at

at

do
im

nd
er

rin

ut
A

co
Pr

lo

O
Se

Ch

Fig. 3 Changes of the mean concentrations of airborne microorganisms in accordance to the changes of solar radiation at
different outdoor stages of a wastewater treatment plant. The wind speed ranged from 0.9 to 3.3 m/s. Error bars indicate
minimum and maximum counts.

(Maier et al., 2000). Additionally, the presence of indicator
microorganisms for faecal contamination in the bioaerosols
was examined by the determination of indicator microorgan-
isms, such as total and faecal coliforms as well as enterococci.
In parallel, detection of fungi was achieved by cultivation of
the impacted bioaerosols in an acidic medium, which
supports the growth of most yeasts and moulds while
inhibiting most bacteria.
As seen in Fig. 1 and Table 2, a gradual decrease of
bioaerosol emissions was observed during the advanced
wastewater treatment from the pretreatment to the primary,
secondary and tertiary treatment. Similar investigations,
using a variety of experimental methodologies, showed a
similar gradual decrease of bioaerosols at wastewater treat-
ment plants (Ranalli et al., 2000; Pascual et al., 2003).
In comparison to similar investigations in wastewater
treatment plants (Ranalli et al., 2000; Stelzenbach, 2002), the
airborne microbes detected in our investigation showed lower
concentrations, of 12 orders of magnitude, even using the air
sampler MAS 100 and by similar wastewater inflow (Pascual
et al., 2003). However, the improvement in the efficiency of
wastewater treatment, such as exchange of coarse bubble
aeration with fine bubble aeration, can lead to a significant
reduction in bioaerosol generation (Fernando and Fedorak,
2005). Although no regulations regarding microbial contam-
ination in the air or bioaerosol concentrations are mandated,
it is generally accepted that the most common practise is the
comparison of airborne microbial concentrations to concen-
trations measured outdoors during the same sampling event
(Stelzenbach, 2002). In this study, the concentrations of

1362
1200
Concentration (CFU/m3); Solar Radiation (W/m2)
1000
800
600
400
200
0 0
10:00
Fig. 4 Concentrations of airborne heterotrophic bacteria and fungi in bioaerosols during a summer day at the sunshine at
the outdoor clean control. The results reported are the average of five duplicates taken at five different days. Error bars
indicate minimum and maximum counts.
airborne microorganisms at the secondary and tertiary
treatments of the wastewater were even lower than in the
outdoor control (Fig. 1 and Table 2). Especially, the concentra-
tions of the indicator microorganisms tended toward zero in
the bioaerosols at the aeration tanks and were not detectable
at the secondary settlers and at the disinfection tanks. These
observations could be a result of an effective processing
wastewater treatment, as well as of the microbial composi-
tion of the wastewater. On the other hand, high temperatures
and solar radiation in the summer could affect the survival of
the aerosolized microbes from the wastewater and result in
partial inactivation. In the present work, we have shown a
slight increase in the airborne bacteria at the wastewater
treatment plant, even of the pathogens, in the late afternoon
and after sunset (Fig. 3). Presumably, this is due to reduced
solar radiation and temperature, allowing greater survival of
the released bacteria. Repair of temporary, lethal, solar
radiation damage may be activated either by longer wave-
lengths of lights, i.e., the so-called photoreactivation repair
(Jagger, 1983), beyond the damaging wavelengths, or sponta-
neously in the dark (so-called dark repair (Freifelder, 1987)).
Thus, in late afternoon, photoreactivation and dark repair
of solar radiation in airborne bacteria (Dimmick, 1960;
Straat et al., 1977) may contribute significantly to the increase
in the airborne population of bacteria at the wastewater
treatment plant. However, the measured concentrations
of the airborne microbes, also after sunset, were more than
an order of magnitude lower than described in existing
literature (Ranalli et al., 2000; Stelzenbach, 2002; Pascual
et al., 2003). Counts of airborne heterotrophic bacteria at a
similar order of magnitude are reported only by Fernando
and Fedorak (2005) at the activated sludge sewage treatment
plant in Edmonton Canada, where improved wastewater
treatment takes place.
ARTICLE IN PRESS
WAT E R R E S E A R C H 41 (2007) 1355 1365
Heterotrophic Bacteria
Fungi 45
Solar Radiation
Temperature (C); Relative Humidity (%)
Temperature (C) 40
Relative Humidity
35
30
25
20
15
10
5
13:00 18:00
Time
The reduction of pathogens during treatment processes,
even with a 12 order of magnitude decrease in bacterial and
viral numbers, cannot exclude the possibility that the actual
concentrations of pathogens can still be significant for effects
on health (Pillai and Ricke, 2002). Moreover, the exact
microbial concentration cannot be determined using only
cultivation-dependent methods, due to the fact that microbes
may be viable but non-cultivable (Wagner et al., 1993; Alvarez
et al., 1995). In addition, aerosolization of microorganisms
and sampling stress can lead to a loss of culture viability
(Marthi et al., 1990; Buttner and Stetzenbach, 1991; Stewart
et al., 1995). Bunger et al. (2000) showed that measuring
cultivable organisms in bioaerosols underestimates the actual
numbers of potentially infectious or allergenic cells or cellular
components among compost workers and can, thereby,
underestimate the risks from toxic or immunopathogenic
effects.
Many environmental factors have been shown to influence
the ability of microorganisms to survive in the air. The most
important of these are relative humidity and temperature.
UV radiation, oxygen content, specific ions, various pollutants
and air-associated factors are also responsible for the loss of
biological activity (Maier et al., 2000). In general, the relative
humidity or the relative water content of the air has been
shown to be of major importance for the survival of airborne
microorganisms (Maier et al., 2000). Bioaerosols released
into the air from water sources (such as, during splashing)
are different from those generated from soil or non-aqueous
surfaces because, in the first case, microorganisms are
usually formed with a thin layer of moisture surrounding
the cells and consist of aggregates of several microorganisms
(Wickman, 1994). The bacterial cells are also found to absorb
water from the atmosphere when the relative humidity
ranges between 20% and 95% (Peccia et al., 2001). Peccia
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 135 5 136 5 1363

A Afternoon sampling Heterotrophic bacteria Morning sampling

16000

Concentration (CFU/m3)
14000
12000 calculated
10000 measured
08000
06000
04000
02000
00000

s
rs

rs
s

s
t

t
en

en
nk

nk
nk

nk
so

so
m

m
a

a
ta

ta
es

es
t

t
at

at
g

g
n

n
oc

oc
re

re
in

in
io

io
pr

pr
et

et
ttl

ttl
at

at
Pr

Pr
er

er
se

se
ge

ge
A

A
ud

ud
y

y
ar

ar
Sl

Sl
im

im
Pr

Pr
Sampling location

B
Afternoon sampling Fungi Morning sampling
4000.00
3500.00
Concentration (CFU/m3)

3000.00
2500.00
2000.00
1500.00
1000.00 calculated
500.00 measured

0.00
s

s
rs

rs
s

s
t

t
en

en
nk

nk
nk

nk
so

so
m

m
ta

ta
ta

ta
es

es
at

at
g

g
n

n
oc

oc
re

re
in

in
io

io
pr

pr
et

et
ttl

ttl
at

at
Pr

Pr
er

er
se

se
ge

ge
A

A
ud

ud
y

y
ar

ar
Sl

Sl
im

im
Pr

Pr

Sampling location

Fig. 5 Comparison of the calculated data using the mathematical model (Pascual et al., 2003) and of the measured
concentrations of (A) heterotrophic bacteria and (B) fungi in bioaerosols at different stages of a wastewater treatment plant
during afternoon (wastewater inflow 16,832 m3/d and wind speed 1.9 m/s) and morning sampling (wastewater inflow
18,065 m3/d and wind speed 2.1 m/s) at two different days.

et al. (2001) have also shown, using cultivation methods and
microscopy, that when aerosolized bacterial cells, such as
Bacillus subtilis, Serratia marcescens, and Mycobacterium sp., are
exposed to a relative humidity exceeding 50%, they tend to
exhibit increased water sorption, which protects the cells
from UV-induced inactivation. Peccia and Hernandez (2001)
have also shown that when aerosolized bacterial cells, such
as Mycobacterium parafortuitum, are exposed to a relative
humidity ranging between 4% and 95%, UV-induced photo-
reactivation protects the cells from UV-induced inactivation.
Measurements of airborne heterotrophic bacteria in an
outdoor clean control area during summer showed a decrease
in their concentration in the air with increasing dosages of
solar radiation. At the same time, the airborne fungi showed
in our study not only a higher resistance to UV irradiation but
also an initial increase of their numbers following by a slight
decrease with increasing dosages of solar radiation (Fig. 4). In
accordance with these results, the expected effects were
obtained at the outdoor facilities of the wastewater treatment
plant by comparable morningafternoon measurements of
the microbial load in the air. An increase in the bacterial
concentration (heterotrophic bacteria, total and faecal coli-
forms as well as enterococci), as well as a decrease in the
fungal concentrations in the air were measured during
afternoon at the outdoor facilities, accompanied by decreas-
ing solar radiation and temperature with no change of the
wastewater inflow. Factors, such as continuous microbial load
from the wastewater into the air, accompanied by decreasing
solar radiation and temperature and increasing relative
humidity are thought to be responsible for these effects.
Authors Lighthart and Schaffer (1994, 1995), by calculating the
bacterial flux from another source (the chaparral) into the
atmosphere, also in mid-summer at a high desert location,
showed that, in the afternoon, the solar radiation damage
 ARTICLE IN PRESS
1364 WAT E R R E S E A R C H
rate decreases until it is less than the repair rate and the new
release rate of bacteria to the atmosphere. Lighthart and
Schaffer (1994) have also shown that the total viable bacterial
concentrations in the atmosphere are very low. Only 0.81% of
a total upward bacterial flux of 76,000 CFU/m2  h from
chaparral into the atmosphere in mid-summer was viable.
The high temperatures, low relative humidity and, particu-
larly, high solar radiation are thought to be the lethal agents
in their experiments.
In this study, the upward bacterial flux from the wastewater
was not measured but the concentration of microorganisms
in the air and their concentration in the wastewater was
compared and very low correlations were found. For example,
the concentration of the measured bacteria in the wastewater
sampled from the aerated grit removal (e.g., total coliforms:
1.6  104 CFU/mL) was, at least, 108 times higher than their
concentration in the air (e.g., total coliforms: 127 CFU/m3).
In the present study, a predictive equation for the
magnitude of bioaerosol release at wastewater treatment
plants (Pascual et al., 2003) was also applied for the
comparison of the measured data with the model calculated
data. Although the use of the bioaerosol release model is a
good tool for predicting of the emission of airborne micro-
organisms at wastewater treatment plants, it could not be
confirmed to be appropriate for the special summer condi-
tions at the current Mediterranean site. According to the
model, the concentrations of bioaerosols depend on the
species, the location, and also the inflow and wind speed at
that location. Solar radiation, temperature and relative
humidity that were found to be significant in our study,
were not included in the model formulation. As observed,
a better correlation between the measured and the
calculated data was achieved by the afternoon sampling,
indicating the possibility of improving the prediction model
by including further meteorological data, such as solar
radiation, temperature and relative humidity, especially for
the summertime.
However, there is still a lack of knowledge on the fate,
distribution and viability of aerosolized microbes, as well as
on the associated health risks for humans and animals. In the
future, health risk models need to be validated using
epidemiological studies that must include both pathogenic
organisms, as well as microbes in combination with allergens,
odorants and other chemicals.
5. Conclusions
In the present work, we have shown that:
 a gradual decrease of bioaerosol emission was observed
during the advanced wastewater treatment, from the
pretreatment to the primary, secondary and tertiary
treatments;
 the highest concentrations of the indicator microorgan-
isms for faecal contamination, measured at the aerated
grit removal (pre-treatment of wastewater), which were, at
least, 96% higher than at the indoor control. In this case,
the associated health risks by long-time exposure of
humans at this location should be evaluated;
41 (2007) 1355 1365
 at the same time, the concentrations of the indicator
microorganisms for faecal contamination at the secondary
and tertiary treatment of the wastewater were, negligible
(tended towards zero or were not detectable);
 a slight increase of the airborne bacteria at the wastewater
treatment plant at the outdoor treatment facilities, even of
the potential pathogens, in the late afternoon and after
sunset, indicating a greater survival of the released
bacteria from the wastewater into the air by low or
inexistent solar radiation;
 in comparison, a decrease of the airborne heterotrophic
bacteria in the air at an outdoor clean control area during
summer was observed by increasing the dosage of solar
radiation;
 the measured and modelled (Pascual et al., 2003) concen-
trations of heterotrophic bacteria and fungi in bioaerosols
at different stages of a wastewater treatment plant during
morning and afternoon sampling lead to the conclusion
that intensive solar radiation and high air temperatures in
the summer are found to be important factors for the non-
survival of microbial load from the wastewater in the air;
 a possibility of improving the used prediction model
(Pascual et al., 2003) is to include further meteorological
data in the equation, such as solar radiation, temperature
and relative humidity, especially for the summertime at a
Mediterranean site (latitude 351 310 north and longitude 241
030 east).
Acknowledgements
Authors want to thank the Municipal Enterprise for Water and
Sewage of Chania (Crete, Greece) (DEYACH) for the permission
of air sampling at the Municipal Wastewater Treatment Plant
of Chania. Especially, we thank the chemical engineers
C. Kotsifaki and her co-workers P. Maraka and G. Pitsoulakis
in the Municipal Wastewater Treatment Plant of Chania for
her kindly help. We would like to thank the meteorologist
Emanuel Lekkas for the supply of the portable instruments
for the measurements of temperature, relative humidity
and wind speed. We also thank the assistant professor
D. Kolokotsa and her co-workers E. Sotiropoulos and
S. Dolianitis at the Laboratory of Renewable Energy Engineer-
ing for the supply of the compass and the solar radiation data.
Assistant professor M. Lazaridis is acknowledged for the
proof reading of the manuscript and his helpful advices.
Finally, we want to thank the two anonymous referees for
their constructive comments and suggestions.
R E F E R E N C E S
Alvarez, A.J., Buttner, M.P., Stetzenbach, L.D., 1995. PCR for
bioaerosol monitoring: sensitivity and environmental inten-
ference. Appl. Environm. Microbiol. 61, 36393644.
American Public Health Association, American Water Works
Association, Water Environment Federation, 1998. Standard
Methods for Examination of Water and Wastewater. 20th ed.,
American Public Health Association, New York, USA.
Bunger, J., Antlauf-Lammers, M., Schulz, T.G., Westphal, G.A.,
Muller, M.M., Ruhnau, P., Hallier, E., 2000. Health complaints
 ARTICLE IN PRESS
WAT E R R E S E A R C H
and immunological markers of exposure to bioaerosols among
biowaste collectors and compost workers. Occup. Environ.
Med. 57, 458464.
Buttner, M.P., Stetzenbach, L.D., 1991. Evaluation of four aero-
biological sampling methods for the retrieval of aerosolized
Pseudomonas syringae. Appl. Environ. Microbiol. 57, 12681270.
Crawford, G.V., Jones, P.H., 1979. Sampling and differentiation
techniques for airborne organisms emitted from wastewater.
Water Res. 13, 393399.
Crowe, J.H., Carpenter, F., Crowe, C.M., Anchordoguy, T.J., 1990. Are
freezing and dehydration similar stress factors? A comparison
of modes 07 interaction of stabilizing solutes with biomole-
cules. Cryobiololgy 21, 9231.
Dimmick, R.L., 1960. Delayed recovery of airborne Serratia
marcescens after short-time exposure to ultra-violet irradia-
tion. Science 4733, 251252.
Fernando, N.L., Fedorak, P.M., 2005. Changes at an activated
sludge sewage treatment plant alter the numbers of airborne
aerobic microorganisms. Water Res. 39, 45974608.
Freifelder, D.M., 1987. Molecular Biology. Jones and Bartlett
Publishers, Inc., Boston.
Gregory, P.H., 1973. Microbiology of the Atmosphere. Wiley, New
York, 377p.
Israeli, E., Schaffer, B.T., Lighthart, B., 1993. Protection of freeze-
dried Escherichia coli by trehalose upon exposure to environ-
mental conditions. Cryobiology 30, 519523.
Jagger, J., 1983. Physiological effects of near-ultraviolet radiation
on bacteria. in: Photochemical and Photobiological Reviews,
vol. 7. Plenum Press, New York, pp. 174.
Lighthart, B., Schaffer, B.T., 1994. Bacterial flux from chapparal
into the atmosphere in mid-summer at a high desert location.
Atmos. Environ. 28, 12671274.
Lighthart, B., Schaffer, B.T., 1995. Airborne bacteria in the atmo-
spheric surface layer: temporal distribution above a grass seed
field. Appl. Environ. Microbiol. 61, 14921496.
Maier, R.M., Pepper, I.L., Gerba, C.P., 2000. Environmental Micro-
biology. Academic Press, San Diego, CA.
Marthi, B., Fieland, V.P., Walter, M., Seidler, R.J., 1990. Survival of
bacteria during aerosolization. Appl. Environ. Microbiol. 56,
34633467.
4 1 (200 7) 135 5 136 5 1365
Merck, 2002. MAS-100 manualmicrobial air monitoring system.
Pascual, L., Perez-Luz, S., Adela Yanez, M., Santamaria, A.,
Gibert, K., Salgot, M., Apraiz, D., Catalan, V., 2003. Bioaerosol
emission from wastewater treatment plants. Aerobiologia 19,
261270.
Peccia, J., Hernandez, M., 2001. Photoreactivation in airborne
Mycobacterium parafortuitum. Appl. Environ. Microbiol. 67,
42254232.
Peccia, J., Werth, H.M., Miller, S., Hernandez, M., 2001. Effects of
relative humidity on the ultraviolet induced inactivation of
airborne bacteria. Aerosol Sci. Technol. 35, 728740.
Pillai, S.D., Ricke, S.C., 2002. Bioaerosols from municipal and
animal wastes: background and contemporary issues. Can.
J. Microbiol. 48, 681696.
Ranalli, G., Principi, P., Sorlini, C., 2000. Bacterial aerosol emission
from wastewater treatment plants: culture methods and bio-
molecular tools. Aerobiologia 16, 3946.
Stelzenbach, L.D., 2002. Introduction to aerobiology. In: Hurst, C.J.,
et al. (Eds.), Manual of Environmental Microbiology. ASM Press,
Washington, DC, pp. 801813.
Stewart, S.L., Grinshpun, S.A., Willeke, K., Terzieva, S., Ulevicius,
V., Donnelly, J., 1995. Effect of impact stress on microbial
recovery on an agar surface. Appl. Environ. Microbiol. 61,
12321239.
Straat, P.A., Wolochow, H., Dimmick, R.L., Chatigny, M.A., 1977.
Evidence of incorporation of thymidine into deoxyribonucleic
acid in airborne bacterial cells. Appl. Environ. Microbiol. 34,
292296.
Wagner, M., Amman, R., Lemmer, H., Schleifer, K.H., 1993. Probing
activated sludge with oligonucleotid specific for Proteobac-
teria: inadequacy of culture-dependent methods for describ-
ing microbial structure. Appl. Environ. Microbiol. 59,
15201525.
Wathes, C., Zaidan, W.A.R., Pearson, G.R., Hinton, M., Todd, N.,
1988. Aerosol infection of calves and mice with Salmonella
typhimurium. Vet. Record 123, 590594.
Wickman, H.H., 1994. Deposition, adhesion and release of
bioaerosols. In: Lighthart, B., Mohr, A.J. (Eds.), Atmospheric
Microbial Aerosols, Theory and Applications. Chapman & Hall,
New York, pp. 99165.