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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


and Ricke, 2002). Many studies have been carried out to
Aerosolization of microbial pathogens, endotoxins, and evaluate the biological risks of aerosols by determining the
odours is an inevitable consequence of the generation and concentrations of viable microorganisms, using different
handling of wastewater. High amounts of microorganisms are sampling and detection methods (Crawford and Jones, 1979;
not only present in the wastewater but also in bioaerosols Buttner and Stetzenbach, 1991; Ranalli et al., 2000; Pillai and
that are generated during the different phases of the process Ricke, 2002; Pascual et al., 2003). However, significant knowl-
in wastewater treatment plants. Bioaerosols can be a vehicle 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
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1356 WAT E R R E S E A R C H 41 (2007) 1355 1365

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

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


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WAT E R R E S E A R C H 4 1 (200 7) 135 5 136 5 1359

removal. At the same time, the mean concentrations of the the decrease of bioaerosol emission from the pretreatment to
indicator microorganisms of faecal contamination at the the sludge processors was 19.7% for the heterotrophic
indoor control (total coliforms: 476 CFU/m3; faecal coliforms: bacteria, 99.4% for the total coliforms, 100% for the faecal
070 CFU/m3; enterococci: 272 CFU/m3; Table 2) were at least coliforms and the enterococci and, finally, 84.2% for the fungi
96% lower than at the aerated grit removal. In comparison, the (Fig. 1 and Table 2).
concentrations of the heterotrophic bacteria, the total coli-
forms, the faecal coliforms, the enterococci, and the fungi in 3.2. Influence of meteorological conditions
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  As mentioned before, sampling of bioaerosols was carried out
1010 CFU/m3), 2  103 CFU/mL (2  109 CFU/m3), 2.5  104 CFU/mL during summer 2005 at a location with latitude 351 310 north
(2.5  1010 CFU/m3), and 7 CFU/mL (7  106 CFU/m3), respec- and longitude 241 030 east, where the solar radiation was
tively. Thus, the concentration of the measured bacteria in intense and the air temperature was high. Fig. 2 shows the
the wastewater was, at least, 108 times higher than the mean hourly meteorological conditions of temperature and
concentration in the air. In contrast, the concentration of the solar radiation during the observation periods. The sampling
measured fungi in the wastewater was 106 times lower than time was usually from 10 a.m. to 3 p.m. when the solar
the concentration in the air. radiation increased roughly from 520 to 840 W/m2. The
A gradual decrease of the concentration of the hetero- ambient temperature in the outdoor facilities ranged
trophic bacteria as well as of the fungi in the bioaerosols was from 25 to 31 1C and the relative humidity at approximately
obtained by advanced treatment of wastewater (secondary 40%. In order to determine the influence of the solar
and tertiary treatment) (Fig. 1). The concentration of the radiation, the temperature and the relative humidity to
heterotrophic bacteria and the fungi decreased more than microorganism concentrations in the air, two sampling
50% after the pretreatment of the wastewater. The concen- campaigns were performed in two different days in the
tration of the airborne microorganisms in the outdoor control morning/noon (from 10 to 3 p.m.) and in the afternoon/
was even higher than those at the secondary and tertiary evening (from 4 to 9 p.m.). During the afternoon/evening
treatments of the wastewater (Fig. 1). In total, 97.4% decrease sampling campaigns the solar radiation decreased from
of the heterotrophic bacteria and 95.8% decrease of the fungi 730 to 10 W/m2, whereas the ambient temperature ranged
was achieved. from 29 to 27 1C and the relative humidity at approximately
Comparable results were obtained also with respect to the 55%. Duplicate samples of bacteria (heterotrophic bacteria,
indicator microorganisms of faecal contamination (Table 2). total coliforms, faecal coliforms and enterococci), as well
In this case, the pathogens tended towards zero in the as of fungi, were collected at each sampling time. The
bioaerosols at the aeration tanks and were not more changes of the mean concentrations of airborne microorgan-
detectable at the secondary settlers or at the disinfection isms from the two compared morning and two afternoon
tanks. A mean concentration of 1 CFU/m3 of total coliforms, campaigns, according to the changes in air temperature,
that was often as high as in the clean outdoor control, was solar radiation and relative humidity at outdoor facilities of
detectable at the aeration tanks. Faecal coliforms and the wastewater treatment plant are summarized in Fig. 3.
enterococci could never be detected in air samples of 2000 L Thereby, an increase in the airborne bacterial concentrations
at the secondary settling tanks or at the disinfection tanks between 2 and 9 times was observed at the outdoor facilities
(Table 2). In comparison, concentrations of 2.4  103 CFU/mL when the ambient temperature and the solar radiation
(2.4  109 CFU/m3), 7  102 CFU/mL (7  108 CFU/m3)and 6.5  decreased. In contrast, a decrease in the airborne fungal
103 CFU/mL (6.5  109 CFU/m3) for the total and faecal coli- concentrations at the wastewater treatment facilities
forms and the enterococci, respectively, were determined in was observed. The fungal concentrations were from 2 to 9
the secondary treated wastewater before disinfection. Thus, a times lower in the afternoon, in comparison to the concen-
total of 100% decrease for the indicator microorganisms of trations at noontime, with the highest solar radiation. In
faecal contamination was achieved. comparison to the data of the outdoor facilities, the concen-
Concentrations of airborne microorganims were also de- trations of the airborne microorganisms at the indoor
termined in the air inside the building with the belt filter facilities were within the expected range.
presses of the waste sludge processors. As shown in Fig. 1, the The effect of the changes in solar radiation, air temperature
mean concentration of the airborne heterotrophic bacteria and relative humidity on the bacterial and fungal counts in
was lower than their concentrations at the pretreatment and the air was assessed by additional measurements at the
primary settlers but higher than at all residual treatment outdoor control. Thereby, the outdoor concentrations of
stages of the wastewater. However, the concentrations of airborne microorganisms were monitored at morning, noon
heterotrophic bacteria at the sludge processors were lower and evening during five different summer days (Fig. 4).
than the concentrations of a busy indoor place used as Measurements of airborne heterotrophic bacteria and fungi
control. In contrast, the counts of the airborne fungi at in the outdoor clean control area at the same time period
the sludge processors were the second highest after their attempted to detect the microbial flux in the air under similar
counts at the grit removal and remarkably higher than environmental conditions. As shown, a decrease in the
the concentrations at the indoor control. Regarding the airborne heterotrophic bacteria concentration from morning
indicator microorganisms, only low concentrations of total to noon was accompanied by an increasing dosage of
coliforms (1 CFU/m3) were measured. Faecal coliforms and solar radiation (at sunshine: 41,640 W  min/m2 at 10 a.m.;
enterococci could not be detected in air samples of 2000 L. So, 170,562 W  min/m2 at 1 p.m.; 352,195 W  min/m2 at 6 p.m.).
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1360 WAT E R R E S E A R C H 41 (2007) 1355 1365

900 35

800
30
700
25
Solar radiation (W/m2)

600

Temperature (C)
500 20

400 15

300
10
200 Solar radiation (W/m2)
Temperature (C)
5
100

0 0
5:00 6:00 7:00 8:00 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

Fig. 2 Mean hourly meteorological conditions of temperature and solar radiation during the observation periods.

Thus, during sunshine, a decrease of bacterial concentrations airborne microbes with advanced wastewater treatment, as
of approximately 7.8 times was achieved between 10 and well as an increase between the concentrations at the
1 p.m. Thereafter, a slower decrease in bacterial concentra- secondary wastewater treatment and at the sludge proces-
tions of approximately 2.4 times between 1 and 6 p.m. with sors, was confirmed in the calculated as well as in the
decreasing solar radiation and temperature, together measured data (Fig. 5). Regarding the concentrations of the
with increasing relative humidity, was observed. In contrast, total and faecal coliforms and enterococci (data not shown),
the airborne fungal concentrations showed a maximum at the slight increasing tendency in the calculated concentra-
noon time (1 p.m.). Thus during sunshine, an increase of tions between the secondary wastewater treatment and the
fungal concentrations of approximately 1.6 times was sludge processors could not be supported by the measured
achieved between 10 and 1 p.m., by increasing the dose of data, probably, due to the low detection of pathogens at these
solar radiation. Finally, with a further increase in the dosage two stages (Table 2).
of solar radiation, a decrease in fungal concentrations of As shown in Fig. 5, the agreement of the measured with the
approximately 1.6 times was obtained, bringing the fungal calculated data regarding the tendency was better during the
concentrations to the morning values (Fig. 4). afternoon sampling than during the morning sampling.
Differences in the meteorological conditions (solar radiation,
3.3. Model comparison temperature and relative humidity) may be responsible
for this.
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 4. Discussion
to correlate the measured and the calculated concentrations
of the airborne microorganisms. However, a low correlation The air sampler MAS 100 is made to comply with ISO/CD
between the calculated data and the measured concentra- 14698-1:1996 recommendations for clean rooms and asso-
tions of airborne bacteria during summer at the Mediterra- ciated controlled environments (Merck, 2002). A number of
nean site under study at different stages of the wastewater cultivation-based approaches are available to detect and
treatment plant was obtained (Fig. 5). The measured data, characterize the viable components of bioaerosols (Marthi
from morning and afternoon samplings, which were taken at et al., 1990; Stewart et al., 1995; Ranalli et al., 2000). In the
different days, differed from the calculated by 12 orders of present study, cultivation-based detection in complete and
magnitude. Additionally, the measured data could not con- selective media directly after impaction was used for the
firm the prediction of the model, that with increasing inflow determination of specific components of bioaerosols emitted
of wastewater, a higher concentration of bioaerosol should during the biological wastewater treatment. Mesophilic
be expected. As shown in Fig. 5, the daily inflow of waste- heterotrophic bacteria were chosen to be analysed because
water from the presented morning sampling was higher of their prevalence in the environment, including among
(18,065 m3/d) than the inflow from the afternoon sampling others bacterial genera such as Pseudomonas, Aeromonas,
(16,832 m3/d). The high calculated microbial concentrations Klebsiella, Bacillus, Staphylococcus, Streptococcus, Escherichia,
during morning sampling were correlated with elevated Enterobacter, Proteus, Alcaligenes, as well as Stenotrophomonas.
wastewater inflow (Fig. 5). However, a gradual decrease of Some members of these genera are opportunistic pathogens
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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 In comparison to similar investigations in wastewater
microorganisms for faecal contamination in the bioaerosols treatment plants (Ranalli et al., 2000; Stelzenbach, 2002), the
was examined by the determination of indicator microorgan- airborne microbes detected in our investigation showed lower
isms, such as total and faecal coliforms as well as enterococci. concentrations, of 12 orders of magnitude, even using the air
In parallel, detection of fungi was achieved by cultivation of sampler MAS 100 and by similar wastewater inflow (Pascual
the impacted bioaerosols in an acidic medium, which et al., 2003). However, the improvement in the efficiency of
supports the growth of most yeasts and moulds while wastewater treatment, such as exchange of coarse bubble
inhibiting most bacteria. aeration with fine bubble aeration, can lead to a significant
As seen in Fig. 1 and Table 2, a gradual decrease of reduction in bioaerosol generation (Fernando and Fedorak,
bioaerosol emissions was observed during the advanced 2005). Although no regulations regarding microbial contam-
wastewater treatment from the pretreatment to the primary, ination in the air or bioaerosol concentrations are mandated,
secondary and tertiary treatment. Similar investigations, it is generally accepted that the most common practise is the
using a variety of experimental methodologies, showed a comparison of airborne microbial concentrations to concen-
similar gradual decrease of bioaerosols at wastewater treat- trations measured outdoors during the same sampling event
ment plants (Ranalli et al., 2000; Pascual et al., 2003). (Stelzenbach, 2002). In this study, the concentrations of
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1362 WAT E R R E S E A R C H 41 (2007) 1355 1365

Heterotrophic Bacteria
1200 Fungi 45

Concentration (CFU/m3); Solar Radiation (W/m2)


Solar Radiation

Temperature (C); Relative Humidity (%)


Temperature (C) 40
1000 Relative Humidity

35

800 30

25
600
20

400 15

10
200
5

0 0
10:00 13:00 18:00
Time

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 The reduction of pathogens during treatment processes,
treatments of the wastewater were even lower than in the even with a 12 order of magnitude decrease in bacterial and
outdoor control (Fig. 1 and Table 2). Especially, the concentra- viral numbers, cannot exclude the possibility that the actual
tions of the indicator microorganisms tended toward zero in concentrations of pathogens can still be significant for effects
the bioaerosols at the aeration tanks and were not detectable on health (Pillai and Ricke, 2002). Moreover, the exact
at the secondary settlers and at the disinfection tanks. These microbial concentration cannot be determined using only
observations could be a result of an effective processing cultivation-dependent methods, due to the fact that microbes
wastewater treatment, as well as of the microbial composi- may be viable but non-cultivable (Wagner et al., 1993; Alvarez
tion of the wastewater. On the other hand, high temperatures et al., 1995). In addition, aerosolization of microorganisms
and solar radiation in the summer could affect the survival of and sampling stress can lead to a loss of culture viability
the aerosolized microbes from the wastewater and result in (Marthi et al., 1990; Buttner and Stetzenbach, 1991; Stewart
partial inactivation. In the present work, we have shown a et al., 1995). Bunger et al. (2000) showed that measuring
slight increase in the airborne bacteria at the wastewater cultivable organisms in bioaerosols underestimates the actual
treatment plant, even of the pathogens, in the late afternoon numbers of potentially infectious or allergenic cells or cellular
and after sunset (Fig. 3). Presumably, this is due to reduced components among compost workers and can, thereby,
solar radiation and temperature, allowing greater survival of underestimate the risks from toxic or immunopathogenic
the released bacteria. Repair of temporary, lethal, solar effects.
radiation damage may be activated either by longer wave- Many environmental factors have been shown to influence
lengths of lights, i.e., the so-called photoreactivation repair the ability of microorganisms to survive in the air. The most
(Jagger, 1983), beyond the damaging wavelengths, or sponta- important of these are relative humidity and temperature.
neously in the dark (so-called dark repair (Freifelder, 1987)). UV radiation, oxygen content, specific ions, various pollutants
Thus, in late afternoon, photoreactivation and dark repair and air-associated factors are also responsible for the loss of
of solar radiation in airborne bacteria (Dimmick, 1960; biological activity (Maier et al., 2000). In general, the relative
Straat et al., 1977) may contribute significantly to the increase humidity or the relative water content of the air has been
in the airborne population of bacteria at the wastewater shown to be of major importance for the survival of airborne
treatment plant. However, the measured concentrations microorganisms (Maier et al., 2000). Bioaerosols released
of the airborne microbes, also after sunset, were more than into the air from water sources (such as, during splashing)
an order of magnitude lower than described in existing are different from those generated from soil or non-aqueous
literature (Ranalli et al., 2000; Stelzenbach, 2002; Pascual surfaces because, in the first case, microorganisms are
et al., 2003). Counts of airborne heterotrophic bacteria at a usually formed with a thin layer of moisture surrounding
similar order of magnitude are reported only by Fernando the cells and consist of aggregates of several microorganisms
and Fedorak (2005) at the activated sludge sewage treatment (Wickman, 1994). The bacterial cells are also found to absorb
plant in Edmonton Canada, where improved wastewater water from the atmosphere when the relative humidity
treatment takes place. ranges between 20% and 95% (Peccia et al., 2001). Peccia
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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 accordance with these results, the expected effects were
microscopy, that when aerosolized bacterial cells, such as obtained at the outdoor facilities of the wastewater treatment
Bacillus subtilis, Serratia marcescens, and Mycobacterium sp., are plant by comparable morningafternoon measurements of
exposed to a relative humidity exceeding 50%, they tend to the microbial load in the air. An increase in the bacterial
exhibit increased water sorption, which protects the cells concentration (heterotrophic bacteria, total and faecal coli-
from UV-induced inactivation. Peccia and Hernandez (2001) forms as well as enterococci), as well as a decrease in the
have also shown that when aerosolized bacterial cells, such fungal concentrations in the air were measured during
as Mycobacterium parafortuitum, are exposed to a relative afternoon at the outdoor facilities, accompanied by decreas-
humidity ranging between 4% and 95%, UV-induced photo- ing solar radiation and temperature with no change of the
reactivation protects the cells from UV-induced inactivation. wastewater inflow. Factors, such as continuous microbial load
Measurements of airborne heterotrophic bacteria in an from the wastewater into the air, accompanied by decreasing
outdoor clean control area during summer showed a decrease solar radiation and temperature and increasing relative
in their concentration in the air with increasing dosages of humidity are thought to be responsible for these effects.
solar radiation. At the same time, the airborne fungi showed Authors Lighthart and Schaffer (1994, 1995), by calculating the
in our study not only a higher resistance to UV irradiation but bacterial flux from another source (the chaparral) into the
also an initial increase of their numbers following by a slight atmosphere, also in mid-summer at a high desert location,
decrease with increasing dosages of solar radiation (Fig. 4). In showed that, in the afternoon, the solar radiation damage
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1364 WAT E R R E S E A R C H 41 (2007) 1355 1365

rate decreases until it is less than the repair rate and the new  at the same time, the concentrations of the indicator
release rate of bacteria to the atmosphere. Lighthart and microorganisms for faecal contamination at the secondary
Schaffer (1994) have also shown that the total viable bacterial and tertiary treatment of the wastewater were, negligible
concentrations in the atmosphere are very low. Only 0.81% of (tended towards zero or were not detectable);
a total upward bacterial flux of 76,000 CFU/m2  h from  a slight increase of the airborne bacteria at the wastewater
chaparral into the atmosphere in mid-summer was viable. treatment plant at the outdoor treatment facilities, even of
The high temperatures, low relative humidity and, particu- the potential pathogens, in the late afternoon and after
larly, high solar radiation are thought to be the lethal agents sunset, indicating a greater survival of the released
in their experiments. bacteria from the wastewater into the air by low or
In this study, the upward bacterial flux from the wastewater inexistent solar radiation;
was not measured but the concentration of microorganisms  in comparison, a decrease of the airborne heterotrophic
in the air and their concentration in the wastewater was bacteria in the air at an outdoor clean control area during
compared and very low correlations were found. For example, summer was observed by increasing the dosage of solar
the concentration of the measured bacteria in the wastewater radiation;
sampled from the aerated grit removal (e.g., total coliforms:  the measured and modelled (Pascual et al., 2003) concen-
1.6  104 CFU/mL) was, at least, 108 times higher than their trations of heterotrophic bacteria and fungi in bioaerosols
concentration in the air (e.g., total coliforms: 127 CFU/m3). at different stages of a wastewater treatment plant during
In the present study, a predictive equation for the morning and afternoon sampling lead to the conclusion
magnitude of bioaerosol release at wastewater treatment that intensive solar radiation and high air temperatures in
plants (Pascual et al., 2003) was also applied for the the summer are found to be important factors for the non-
comparison of the measured data with the model calculated survival of microbial load from the wastewater in the air;
data. Although the use of the bioaerosol release model is a  a possibility of improving the used prediction model
good tool for predicting of the emission of airborne micro- (Pascual et al., 2003) is to include further meteorological
organisms at wastewater treatment plants, it could not be data in the equation, such as solar radiation, temperature
confirmed to be appropriate for the special summer condi- and relative humidity, especially for the summertime at a
tions at the current Mediterranean site. According to the Mediterranean site (latitude 351 310 north and longitude 241
model, the concentrations of bioaerosols depend on the 030 east).
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, Acknowledgements
were not included in the model formulation. As observed,
a better correlation between the measured and the Authors want to thank the Municipal Enterprise for Water and
calculated data was achieved by the afternoon sampling, Sewage of Chania (Crete, Greece) (DEYACH) for the permission
indicating the possibility of improving the prediction model of air sampling at the Municipal Wastewater Treatment Plant
by including further meteorological data, such as solar of Chania. Especially, we thank the chemical engineers
radiation, temperature and relative humidity, especially for C. Kotsifaki and her co-workers P. Maraka and G. Pitsoulakis
the summertime. in the Municipal Wastewater Treatment Plant of Chania for
However, there is still a lack of knowledge on the fate, her kindly help. We would like to thank the meteorologist
distribution and viability of aerosolized microbes, as well as Emanuel Lekkas for the supply of the portable instruments
on the associated health risks for humans and animals. In the for the measurements of temperature, relative humidity
future, health risk models need to be validated using and wind speed. We also thank the assistant professor
epidemiological studies that must include both pathogenic D. Kolokotsa and her co-workers E. Sotiropoulos and
organisms, as well as microbes in combination with allergens, S. Dolianitis at the Laboratory of Renewable Energy Engineer-
odorants and other chemicals. 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.
5. Conclusions Finally, we want to thank the two anonymous referees for
their constructive comments and suggestions.
In the present work, we have shown that:
R E F E R E N C E S
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