429

Anna Gotkowska-Pachta1
Zofia Filipkowska1
Ewa Korzeniewska1
Wojciech Janczukowicz2
Beverly Dixon3
Iwona Goas1
Damian Szwalgin1
1
Faculty of Environmental Sciences,
Department of Environmental
Microbiology, University of Warmia
and Mazury in Olsztyn, Olsztyn
Kortowo, Poland
2
Faculty of Environmental Sciences
and Fisheries, Department of
Environment Protection Engineering,
University of Warmia and Mazury in
Olsztyn, OlsztynKortowo, Poland
3
Department of Biological Sciences,
California State University, Hayward,
CA, USA
Research Article
Airborne Microorganisms Emitted from Wastewater
Treatment Plant Treating Domestic Wastewater and
Meat Processing Industry Wastes
Experiments were conducted to study the airborne microbial contamination generated
by a wastewater treatment plant (WWTP). Aerosol samples were collected simul-
taneously, by sedimentation and impact methods, from the area and the surroundings
of the WWTP. Total colony forming units (CFUs) of heterotrophic bacteria (HPC), as well
as members of the Enterobacteriaceae, staphylococci, enterococci, actinomycetes, and
microscopic fungi were determined. Bacterial (HPC) concentrations ranged between 101
and 104 CFU/m3, fungi 0 and 104 CFU/m3. Higher numbers of HPC bacteria in air samples
were observed in summer, fungi in autumn. The main emission of microorganisms to
atmospheric air was from the mechanical sewage treatment devices of the WWTP. The
facilities of the biological sewage treatment of the plant did not generate large amounts
of bioaerosols. In the air obtained from the premises of the WWTP, 25 species of the
Enterobacteriaceae were isolated (Salmonella spp., Klebsiella pneumoniae, Escherichia coli). At
the fence and in the surroundings only Pantoea spp. were identified. This suggests that
the sewage bacteria were mainly discharged in the area of the WWTP. The presence of
enteric bacteria, especially Enterobacteriaceae reflects the level of air pollution with
bioaerosols from sewage and is an important factor during monitoring the quality
of the air around WWTPs.
Keywords: Bioaerosol; Enteric bacteria; Microscopic fungi; Sewage; Wastewater treatment plant
Received: September 6, 2011; revised: January 19, 2012; accepted: April 27, 2012
DOI: 10.1002/clen.201100466

1 Introduction features, the time of the day, the season, and the type of treatment
technology [9, 1521]. Aerosols containing pathogenic micro-
In recent years the number of wastewater treatment plants (WWTPs)
organisms generated by the technological facilities at WWTPs can
has increased worldwide. As a rule, such facilities are located outside
be dispersed over considerable distances, producing adverse effects
urban settlements, but as towns grow, WWTPs are increasingly
on living organisms, primarily by inhalation.
found very close to urbanized areas [1]. Pollutants emitted during
The main objective of this study was to analyze the bacterial
the process of sewage and wastewater treatment (odors, bioaerosols,
(especially from Enterobacteriaceae) and fungal contamination of
chemicals) can be harmful to human and animal life [25]. Biological
atmospheric air on the premises and in the surroundings of the
aerosols produced during wastewater treatment (discharging,
WWTP, which treated predominantly domestic sewage and waste-
mixing, aerating, and spraying of sewage) can contain various
water from the meat processing industry. The presence of these
pathogenic microbes, such as viruses, fungi and bacteria, especially
microorganisms and their identification reflect the level of air
intestinal bacteria from the Enterobacteriaceae [611]. Some of
pollution with bioaerosols from sewage, and are a vital consider-
these microorganisms are associated with toxic pneumonia
ation for monitoring the air quality around WWTPs. Wastewater
(inhalation fever, organic dust toxic syndrome ODTS), chronic
treatment using activated sludge and fine bubble aeration
bronchitis, asthma, and other disorders [2, 3, 8, 1114]. The dispersal
utilized at this WWTP, is one of the most commonly used techno-
of bioaerosols outside the facilities of WWTPs, as well as the
logies [16, 19, 20, 22]. Therefore, the results may serve as a good
type, amount and survival rate of airborne microorganisms depend
comparison with other sites using the same type of technology.
on physicochemical and meteorological air conditions, landscape

2 Materials and methods

Correspondence: Dr. A. Gotkowska-Plachta, Faculty of Environmental
/
Sciences, Department of Environmental Microbiology, University of
Warmia and Mazury in Olsztyn, R. Prawochenskiego Street 1, 10957
OlsztynKortowo, Poland
E-mail: aniagp@uwm.edu.pl
Abbreviations: CFU, colony forming unit; HPC, heterotrophic bacteria;
WWTP, wastewater treatment plant
2.1 Area of study
The wastewater treatment plant in Ostroda in the north-east
of Poland was refurbished in 20012002. Its current capacity is
7000 m3 sewage/day. The plant receives domestic sewage (80%) and
sewage from the meat processing industry (20%). Sewage are purified
mechanically and biologically (activated sludge technology). At this

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430 A. Gotkowska-Pachta et al.

seven sites located (grate chamber, grit chamber, retention chamber,

Figure 1. Technological scheme of the WWTP and the  sampling site
location:  C: control site; Mechanical treatment (1: grate chamber, 2: grit
chamber, 3: retention chamber, 4: preliminary settling tank); Biological
treatment (5: pre-denitrification tank, 6: nitrification and denitrification
tanks, 7: secondary sedimentation tank); Surrounding (8: fence, 9: 50 m,
10: 100 m, 11: 200 m from the fence); A: sludge field.
plant the aeration chamber is equipped with a fine bubble deep
aeration system. The treated wastewater are discharged by under-
ground sanitary collector (5.6 km) to the Drweca River. Individual
mechanical and biological facilities of studied plant are shown in
Fig. 1.
2.2 Air sample collection
The air samples on the premises and in the surroundings of the
WWTP were collected by two methods simultaneously: sedimen-
tation and impact. To study air quality, 12 sampling sites were
chosen. The control site (C), the background was always situated
about 500 m to the windward side. In the WWTPs area there were
preliminary settling tank, pre-denitrification tank, nitrification, and
denitrification tanks, secondary sedimentation tank) and four ones
outside (at the fence of the plant, and 50, 100, and 200 m from the
fence; Fig. 1). All samples were taken downwind (approximately 1
1.5 m from the source). Collection of atmospheric air samples,
according to Polish standards [23, 24] was carried out in two annual
cycles (in 2005 and 2006) in spring, summer, autumn, and winter.
Aerosol samples were collected from 9 am to 3 pm, from all plant
sites and the surroundings. For both methods, Petri dishes contain-
ing appropriate sterile medium were exposed to air on the table at
1.30 m height. In the sedimentation method for HPC and fungi
exposed time was 10 min and for selected medium 30 min according
to Polish standards [23, 24]. In the impact method, samples were
collected by means of an agar impact sampler surface air system
MAS-100 Eco Merck with 400 holes. The impact sampler has a flow
rate of 100 L air/min. The air was aspirated onto a 90 mm contact dish
containing appropriate agar medium. The impaction speed of
the airborne microorganisms on the agar surface was 11 m/s. Air
sampler has the option to regulate the air intake in the range from 1
to 1000 L (the number of liters is set experimentally and depends on
the expected air pollution, the season of the year and the type
of determined microorganisms). In our research, after preliminary
studies, we defined the following volumes of air sampling: 2
102
(in spring, summer, autumn) and 3
102 m3 (in winter) for HPC and
fungi. For other groups of bacteria was 6
102 (in spring, summer,
autumn) and 10
102 m3 (in winter; Tab. 1). At each sampling site,
during four research seasons, three air samples were collected by
sedimentation method and other three samples by impact method,
which gives 24 air samples for each group of microorganisms.
In total, 2016 air samples were collected. For each method, sampling
of microbial parameters was carried out in triplicate. The samples
were transported to the laboratory in refrigerated boxes within 35 h
after sampling. After the incubation, all results of microbiological
measurements were calculated (according to Polish standards) as
CFUs per cubic meter of air (CFU/m3).
2.3 Microbiological analyses
The microbiological studies of air samples involved isolation, identi-
fication, and enumeration of seven groups of microorganisms. The

Table 1. The composition of media used in the microbiological analyses, conditions of air samples collection, and conditions of microorganisms incubation

Group of bacteria Composition (g/100 mL) Temperature of Time of Timea) of agar Volume
of media used in incubation (8C) incubation dishes exposure (
102 m3) of
microbiological analyses (min) (sedimentation air samplesb)
method) (impact method)

Heterotrophic bacteria (HPC) Nutrient agarc) 26 72 h 10 23

Enterobacteriaceae Chromocult coliform agarc) 37 24 h 30 610
Endo agarc) 37 24 h
Enterococci Membrane-filter enterococcus selective 37 72 h 30 610
agar acc. to Slanetz and Bartleyc)
Staphylococci Chapman agarc) 37 48 h 30 610
Pseudomonas fluorescens King agar Bc) 26 48 h 30 610
Actinomycetes Pochon mediuma) 26 7 days 30 610
Fungi (molds, yeasts RBC medium (rose-bengal 25  3 37 days 10 23
and yeast-like fungi) chloramphenicol agar) c)
a)
References: Polish standards [24].
b)
Depends on season: in spring, summer, autumn: 26; in winter 310.
c)
Merck Poland.

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 Airborne Microorganisms Emitted from Wastewater Treatment Plant 431

detailed information about isolated microorganisms, conditions of
their incubation, and composition of media used in the microbio-
logical analyses is described in Tab. 1.
The occurrence of Pseudomonas fluorescens was verified under the
light of the UV lamp (wavelength 365 nm); all colonies which pro-
duced fluorescein were counted and identification confirmed with
API NE multitest strips (bioMerieux) at temperature of incubation
29  28C.
All typical Enterobacteriaceae colonies grown on Endo and
Chromocult media, and staphylococci on Chapmans medium were
inoculated onto the agar-bullion medium with 7% sheep blood
added to enhance bacterial growth and to detect hemolysins.
Additionally, all bacteria grown on Endo and Chromocult media
were analyzed for the presence of cytochrome oxidase (using 1%
N,N,N,N-tetramethyl-p-phenylene-diamine dihydrochloride solution),
and oxidase-negative strains were identified with API 20E multitest
strips (bioMerieux). Representative colonies: salmon to red, dark-
blue to violet, light-blue to turquise colony from Chromocult
medium (173) and each colony from Endo medium (80) were
chosen. In addition to testing for hemolysins, staphylococci
were analyzed for the production of catalase (using 3% hydrogen
peroxide solution) and coagulase production using lyophilized rab-
bit plasma with EDTA. Final identification (each grown colony 74)
was carried out with API STAPH multitest strips (bioMerieux).
Prior to biochemical identification, all isolates were stained by the
Gram method. Identification of fungi was determined by macro- and
micro-morphological characteristics, using standard taxonomic keys
and available literature [25, 26].
The yeasts and yeast-like fungi were identified (each grown
colony 90) with API 20 C AUX multitest strips (bioMerieux).
2.4 Meteorological observation
The meteorological conditions like air temperature and humidity were
measured by electronic humidity/temperature Loggerer EBI2-TH-611/
6120. Wind speed was measured by the Anemometer Skywatch Meteos,
Switzerland and wind direction by a small flag set on the area at
the height of the anemometers. All meteorological parameters
were recorded parallel to the sample collecting for microbiological
analyses.
2.5 Statistical analysis
The one-way analysis of variance (ANOVA) was used to evaluate whether
the number of studied groups of bacteria detected in the air samples
were dependent on the method, time, and places of sample collecting.
Estimation by Spearmans correlation between numbers of studied
microorganisms and meteorological data were used in this study. The
tests were performed with the software STATISTICA 8 (StatSoft Poland).
3 Results and discussion
3.1 Environmental parameters
During the study, the speed of winds (m/s) in particular seasons of
the year was varied: in spring 2.8  0.3, in summer 1.5  0.2, in
autumn 2.0  1.0, and in winter 2.5  1.5. Temperature (8C) varied:
in spring 23.1  1.6, in summer 28.8  1.8, in autumn 22.8  1.5, and
in winter 3.0  1.2. The relative air humidity was as follows: in spring
46.1  3.8%, in summer 65.1  9.4%, in autumn 58.0  11.2%, and in
winter 60.5  18.6%.
3.2 Microbiological parameters at the control site
The southwest wind was predominant during the entire study. The
control site and sampling posts were designated in the same places
of the plant, to the windward side. The number of HPC collected at
these posts ranged from 5.9
101 to 1.1
103 CFU/m3. The examined
air contained Pantoea sp.3 up to 1.1
102 CFU/m3 (Tabs. and 3). Other
bacteria, the Enterobacteriaceae, staphylococci, and enterococci were
not detected. Actinomycetes were present up to 2.0
102, yeasts up
to 2.5
102, and molds up to 3.9
103 CFU/m3. The dominant molds
at the control site were Actinomucor, Alternaria, Aspergillus, Chaetomium,
Chrysosporium, Cladosporium, Cunninghamella, Diplosporium, Fusarium,
Geotrichum, Mucor, Penicillium, Phoma, Rhizopus, Scopulariopsis, Sporothrix,
Thamnidium, Trichoderma, Trichothecium (Tabs. 2 and 4).

Table 2. Median (med.), minimum (min.), and maximum (max.) microorganisms levels (CFU/m3)a) in the air samples in the WWTPs premises and
surroundings, during the whole time of the study

Variable Control site n 24 Mechanical treatmentb) n 96 Biological treatmentc) n 72 Surroundingsd) n 96

Med. Min./max. Med. Min./max. Med. Min./max. Med. Min./max.

2 1 3 2 1 4 2 1 3 2
Heterotrophic bacteria 8.9
10 5.9
10 /1.1
10 8.8
10 0.8
10 /1.2
10 3.5
10 0.8
10 /4.8
10 2.9
10 0.8
101/6.5
103
(HPC)
Enterobacteriaceae 0 0/0 0.3
101 0/1.5
103 0 0/2.9
101 0 0/3.6
101
(Endo medium)
Enterobacteriaceae 0 0/1.1
102 7.6
101 0/2.3
103 1.9
101 0/8.0
102 0 0/2.1
102
(Chromocult medium)
Enterococci 0 0/0 0 0/1.2
102 0 0/2.9
101 0 0/1.0
101
Staphylococci 0 0/0 0 0/2.5
101 0 0/2.9
101 0 0/2.0
101
Actinomycetes 2.5
101 0/2.0
102 1.4
101 0/4.4
102 0.9
101 0/2.9
102 1.0
101 0/3.6
102
Molds 2.5
102 0/3.9
103 1.9
103 0/1.4
104 5.1
102 0/1.4
104 5.2
102 0/2.1
104
Yeasts 6.3
101 1.7
101/2.5
102 4.4
101 0/4.0
102 0 0/9.7
102 0 0/5.0
102
a)
Colony forming units per cubic meter of air.
b)
Grate chamber (site 1), grit chamber (site 2), retention chamber (site 3), preliminary settling tank (site 4).
c)
Pre-denitrification tank (site 5), nitrification and denitrification tanks (site 6), secondary sedimentation tank (site 7).
d)
Fence (site 8), 50 m (site 9), 100 m (site 10), 200 m (site 11) from the fence of the WWTP.
n, the number of air samples taken by the sedimentation and impact methods, for one group of microorganisms at each sampling area in
the whole period of the study.

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432 A. Gotkowska-Pachta et al.

Table 3. Enterobacteriaceae bacteria identified with API 20E tests in the air samples collected in the WWTPs area and its surroundings

Site Bacteria identified

Control site WWTP area Pantoea spp. 3a)

Grate chamber Enterobacter sakazakii
Grit chamber Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Enterobacter amnigenus, Enterobacter cloacae,
Escherichia coli, Escherichia coli 1, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella pneumoniae,
Klebsiella terrigena, Kluyvera spp., Providencia alcalifaciens/rustigianii, Serratia ficaria, Serratia liquefaciens
Retention chamber Citobacter freundii, Citobacter spp., Enterobacter cloacae, Enterobacter amnigenus, Escherichia coli,
Klebsiella ornithinolytica, Pantoea spp. 3
Preliminary settling tank Enterobacter cloacae, Escherichia coli 1, Klebsiella pneumoniae, Klebsiella terrigena, Pantoea spp. 2,
Pantoea spp. 3, Providencia spp., Serratia liquefaciens, Salmonella spp.
Predenitrification tank Citobacter youngae, Escherichia coli 1, Escherichia coli 3, Enterobacter cloacae, Kluyvera spp.,
Serratia rubidea, Salmonella spp.
Nitrification and denitrification tanks Enterobacter aerogenes, Serratia liquefaciens, Pantoea spp. 2
Secondary sedimentation tank Pantoea spp. 2
Surroundings
Fence Pantoea spp. 3
50 m Pantoea spp. 2
100 m Pantoea spp. 3
200 m Pantoea spp. 2
a)
1, 2, 3: different biotypes within a species.

3.3 Quantitative and qualitative composition of the median concentrations of HPC was 8.8
102 CFU/m3. It was
similar to these collected at the control site (8.9
102 CFU/m3),
microorganisms in the air samples collected from
but higher than those collected near the facilities of biological
the area and surroundings of the WWTP sewage treatment of the plant (3.5
102 CFU/m3) and its surround-
In the area and the surroundings of the WWTP 2016 air samples were ings (2.9
102 CFU/m3; Tab. 2).
analyzed. The counts of HPC found in the air samples in the WWTPs The bacterial counts of fecal bacteria from Enterobacteriaceae
area were between 0.8
101 and 1.2
104 CFU/m3. In the mechanical ranged from 0 to 2.3
103 CFU/m3 in the samples of air obtained
sewage treatment devices (the grate chamber and the grit chamber) in the mechanical sewage treatment devices and from 0 to 8.0
102
near the facilities of biological sewage treatment (Tab. 2).
Higher numbers of enteric bacteria in the air samples at the
Table 4. The occurrence of molds in studied air samples in the WWTPs mechanical sewage treatment devices (the grate chamber and the
area and its surrounding grit chamber) of the plant could have been caused by the constant
flow of raw wastewater through the aerated grit chamber, in which
Genus of molds WWTP area WWTP surrounding small droplets of bioaerosols were produced and dispersed to the air
Absidia 1, 2 9 by wind. Another factor which may have raised the microbial counts
Actinomucor C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10 at these sampling sites, was a worm wheel conveyor, periodically
Alternaria C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11 switched on to carry sand from the grit chamber to a nearby con-
Aspergillus C, 1, 2, 3, 4, 5, 6 8, 9, 10, 11 tainer. At these times, even a light wind could have dispersed large
Botrytis 7 11
quantities of microorganisms.
Chaetomium C, 5
Chrysosporium C, 2, 3, 5, 7 8, 9, 10, 11 The level and range of bioaerosols emitted to the air from WWTPs
Cladosporium C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11 depends on the type of technology employed for wastewater treat-
Cunninghamella C, 6 ment and aeration system [1517, 2022, 27]. Microorganism emis-
Diplosporium C, 4 sion is lower when depth aeration, rather than surface, is employed.
Doratomyces 9
Fusarium C, 5 8 In the examined sewage treatment plant the aeration chamber was
Geotrichum C, 1, 2, 3, 5, 6, 7 8, 9, 10 equipped with a fine bubble deep aeration system which did not
Gliocladium 8 produce large turbulence, and consequently did not generate a large
Mucor C, 1, 2, 3, 4, 5, 6, 7 8, 9, 11 amount of bioaerosols. These results correspond with the results of
Nigrospora 6
other authors conducting research in similar operational conditions
Penicillium C, 1, 2, 5, 7 8, 10
Phoma C [9, 20, 28, 29]. When surface aeration was employed, the emission of
Rhizopus C, 1, 3 8 microorganisms in the areas adjacent to aeration chambers was the
Scopulariopsis C, 2, 3, 4, 6 8 highest [15, 20].
Sporothrix C In the air samples obtained on the premises of the WWTP,
Thamnidium C, 2, 3
Trichoderma C, 4, 5 8 165 identified strains belonging to 25 bacteria species from
Trichothecium C, 4, 5, 6 10 Enterobacteriaceae were isolated. At the fence and in the immediate
surroundings of the plant up to 200 m, only the species belonging
C: control site, WWTPs area (1: grate chamber, 2: grit chamber, 3: to the genus Pantoea were identified (Tab. 3). The Pantoea bacteria
retention chamber, 4: preliminary settling tank, 5: predenitrifica-
are widely distributed in nature and isolated from numerous eco-
tion tank, 6: nitrification and denitrification tanks, 7: secondary
sedimentation tank), surroundings (8: fence, 9: 50 m, 10: 100 m, logical niches, including plants, water, soil, humans, and animals.
11: 200 m). The presence of Pantoea bacteria in the surroundings of the WWTP

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 Airborne Microorganisms Emitted from Wastewater Treatment Plant
may have been related to its relatively high abundance in the bio-
aerosol sampled at the area of the investigated plant, where they
accounted for 20.5% of all identified strains. It could also be due to
higher radiotolerance caused by the presence of yellow carotenoid
pigment [30]. It is known that even a small concentration of caroten-
oid plays a beneficial role in the resistance to radiation [31, 32].
Pantoea bacterium is closely related to Escherichia coli, but is up to five
times more radiotolerant [33].
The highest diversity of bacteria (21 species including Salmonella
spp.) isolated from the air sampled near mechanical sewage treat-
ment devices (15 species near grit chamber), indicates that the
qualitative composition of bacteria may have been affected by waste-
water from meat processing. The microbiota of wastewater is as
varied as the composition of pollutants. The highest amounts and
diversity of microorganisms are found in domestic sewage along
with human and animal excreta, which may include bacteria:
Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Serratia,
Staphylococcus, Salmonella, Shigella, and Vibrio [6, 10, 19, 21].
However, it is not easy to delineate whether differences in bioaerosol
emissions are related to the type and amount of treated wastewater,
or to the processes utilized by the plants.
Another enteric bacteria enterococci were mainly identified
(up to 1.2
102) in the air sampled in the WWTPs area
(grate chamber, grit chamber, preliminary settling tank), and
sporadically in negligible numbers in the biological part of the
plant (Tab. 2).
The number of staphylococci in the sampled air did not
exceed 2.9
101 CFU/m3 (Tab. 2). Many authors [6, 10, 19, 21] have
reported that staphylococci were of minor concern in the sewage
and in the air in the WWTPs area. In the air of the studied plant,
15 strains of staphylococci were identified (Staphylococcus haemolyticus,
S. epidermidis, S. lentus, S. xylosus, S. cohni, S. capitis). S. lentus and
S. xylosus were predominant in the air on the plant. On the other
hand, in the air of the Bologna sewage plant, De Luca et al. [34]
recovered 13 species of coagulase-negative staphylococci. Most
common were S. haemolyticus, S. xylosus, and S. cohnii.
P. fluorescens, a typical water/soil microorganism [35], was not
observed in the air samples from the premises or surroundings of
the WWTP.
Actinomycetes, indicating soil borne contamination [36], were
found in counts ranging from 0 to 4.4
102 CFU/m3 (Tab. 2).
These microorganisms were detected on the WWTPs premises
(median 1.4
101), in the surroundings (median 1.0
101), and
at the control sites (median 2.5
101).
The results of mycological analyses of air samples collected in
the area of the WWTP, as well as outside, demonstrated a higher
number of molds (from 0 to 2.1
104 CFU/m3) than yeasts (from 0
to 9.7
102 CFU/m3; Tab. 2). On the WWTPs premises the most
abundant yeasts were Candida and Cryptococcus, in the WWTPs
immediate surroundings Candida and Rhodotorula were isolated
sporadically.
The air samples of WWTPs area and surroundings, contained
molds from genera Actinomucor, Alternaria, Aspergillus, Chrysosporium,
Cladosporium, Geotrichum, Mucor, Penicillium at most sites (Tab. 4).
Among airborne fungi, Penicillium, Aspergillus, Alternaria, and
Cladosporium strains have the greatest potential to evoke allergic
reactions [37]. The most abundant fungi in the atmosphere
(Cladosporium, Penicillium, and Aspergillus) produce high numbers of
small and light spores, and this certainly favors their dominance
in this environment [38].
2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com
433
Soil and plants are probably the main source of molds in the
air of WWTPs surroundings. Yeasts occurred sporadically in air
samples from the WWTPs surroundings. This may be due to larger
size of their cells in comparison to the mold spores which makes
it difficult to transfer them with bioaerosol. However, these micro-
organisms are reported in greater amounts in wastewater (up to 104),
therefore, they should be regarded as microorganisms related with
sewage [1].
The results of the microbiological assays of the atmospheric air
samples showed that the dominant microorganisms in atmospheric
air, both on the WTTPs property and in its immediate surroundings
(to 50 m from the fence), were molds common in the natural
environment. Similar results were obtained by Korzeniewska [1],
Korzeniewska et al. [21], Kazmierczuk et al. [27].
Generally, the higher amounts of analyzed groups of microorgan-
isms were observed in the air sampled near the mechanical sewage
treatment devices (the grate chamber and the grit chamber) than
that sampled near the facilities of biological sewage treatment (the
pre-denitrification tank, the nitrification, and denitrification tanks,
the secondary sedimentation tank; Tab. 2).
However, statistical analysis of research results did not confirm
statistically significant ( p > 0.05) differences in the number of inves-
tigated microorganisms (except Enterobacteriaceae, isolated on
Chromocult medium p < 0.0003 and enterococci p < 0.003) at
different sampling sites.
Taking into account the sampling method, usually higher
amounts of investigated microorganisms were observed in air
samples collected by sedimentation method.
Statistical analysis of research results confirmed statistically
significant differences between the counts: HPC ( p 0.0054);
Enterobacteriaceae (on Chromocult medium p 0.038, and on Endo
medium p 0.017); actinomycetes ( p 0.033) in the air samples
collected by both the sedimentation and impact methods (Fig. 2).
Higher counts of microorganisms in air samples collected by
sedimentation result from the fact that microorganisms sediment
on medium for 10 or 30 min in accordance to Polish standards
[23, 24]. In the impact method some of microorganisms were swept
away by a strong air stream produced by the air sampler, which
meant that some of them could not settle on the medium placed in
the sampler [27]. The air being sucked in or pushed out by volumetric
air samplers can disturb the surrounding area, because it remains in
the area being checked, producing an artificial turbulence, and
thus altering the counts. This kind of method of air samples
collecting, however, is simple, easy to use and reproducible as it
enables researchers to collect a certain volume of air and to obtain
uniform growth and development of microbial colonies on the
surface of a dish [39]. The particulate in bioaerosol sampled by
MAS-100 is 1.12.1 mm in diameter. This is the part of respirable
fraction which may penetrate into the lower respiratory tract posing
risk to human health.
3.4 Seasonal variation
The average counts of microorganisms determined tended to be the
highest in the air samples collected in spring for staphylococci, in
summer for HPC, in autumn for actinomycetes and fungi, and in
winter for Enterobacteriaceae. Statistical analysis confirmed statisti-
cally significant differences in the number of investigated micro-
organisms (except for Enterobacteriaceae isolated on Endo medium
and enterococci) depending on the sampling season (Fig. 3).
Clean Soil, Air, Water 2013, 41 (5), 429436
434 A. Gotkowska-Pachta et al.

Figure 2. Average numbers (CFU/m3) of (A) het-

erotrophic bacteria (HPC), (B) Actinomycetes,
(C) Enterobacteriaceae on Chromocult medium,
(D) Enterobacteriaceae on Endo medium, from
air samples collected by different methods (S,
sedimentation; I, impact). Independent variable
(assembling): method. RMS  random mean
square; N, number of samples; p, significance
level.

Figure 3. Average numbers (CFU/m3) of (A) het-

erotrophic bacteria (HPC), (B) Enterobacteriaceae
on Chromocult, (C) Staphylococci, (D)
Actinomycetes, (E) Molds, (F) Yeasts, from air
samples collected at the different sites during
the whole season of study. Independent variable
(assembling): season. RMS  random mean
square; N, number of samples; p, significance
level.

2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean Soil, Air, Water 2013, 41 (5), 429436
 Airborne Microorganisms Emitted from Wastewater Treatment Plant 435

As Jones and Harrison [40], Korzeniewska [1], Agranovski et al. [41]
suggested, the seasonal variations of bacterial load might be depen-
dent on local meteorological conditions (wind speed and direction,
humidity, temperature, UV radiation), pollutants, and the intrinsic
sensitiveness of different bacteria genera to these factors.
In our study during the spring, when measured humidity was the
lowest (46.1  3.8%) and temperature was 23.1  1.68C, the highest
number of staphylococci in the air samples was determined. It was
confirmed statistically by a negative correlation between the num-
ber of staphylococci (r 0.286, p 0.006) and air humidity (Tab. 5).
These hazardous microorganisms can survive in environment in
the conditions unfavorable for other bacteria. De Luca et al. [34] and
Makison and Swan [42] reported the presence of coagulase negative
staphylococci in different environments (in atmospheric air and on
hard hospital surfaces) with low humidity. These bacteria may occur
as agglomerations of cells, or may be rafted into the air on plant or
animal fragments, on soil particles [40].
As our study shows, the highest amounts of HPC bacteria in the
summer, compared with other research seasons, were observed at
the air temperature of (28.8  1.88C; Fig. 3). It was confirmed by a
significant positive correlation (r 0.384, p 0.0002) between HPC
bacteria and air temperature (Tab. 5). In the summer, these bacteria
had the most favorable conditions to multiply, therefore their con-
centration in wastewater and emitted bioaerosols could have
increased.
In autumn, the counts of molds and actinomycetes were the
highest, particularly in the air samples taken from the sites outside
the fence of plant, and from the control site (Fig. 3). There was
a statistically significant positive correlation noted between the
number of these microorganisms and the temperature: for actino-
mycetes (r 0.321, p 0.002) and for molds (r 0.528, p < 0.001)
(Tab. 5). Relatively high air temperatures (22.8  1.58C) noted during
that season affected their growth. As Kaarakainen et al. [43],
Korzeniewska [1] and Korzeniewska et al. [21] observed, higher
amounts of actinomycetes and fungi were determined in the air
of municipal facilities in autumn. Increased number of actinomy-
cetes, which are indicators of soil contamination [36], may have been
associated with more intensive farming practice during that time of
the year. The increased mold growth may have been enhanced by
dying and decomposing vegetation in the surroundings of the
WWTP during autumn.
Table 5. Statistical estimation by Spearmans rank correlation between
numbers (CFU/m3)a) of studied microorganisms isolated from the air during
the whole time of study and some meteorological parameters
During the winter air sampling there was thick snow cover on the
ground and around the WWTP, which prevented microorganisms
on the surface of soil from entering the atmospheric air. This
means that the microorganisms (from Enterobacteriaceae) found in
the air at that time had been derived from the technical facilities of
the plant. In the winter, the highest amounts of Enterobacteriaceae
in sampled air were observed (Fig. 3). There was also noted the
highest air humidity (60.5  18.6%), which was significantly positively
correlated to the abundance of investigated Enterobacteriaceae
(r 0.217, p 0.041; Tab. 5). Temperature difference between treated
wastewater and ambient air caused increased evaporation of
sewage and condensation of microorganisms. The result of this
was increased emission of enteric bacteria to the air.
In our study there were no statistically significant correlations
between the concentration of determined microorganisms in the
air at designated sampling sites and wind speed observed (Tab. 5).
It may be due to the fact that throughout the study period the
wind was blowing at low speed, ranging from 1.5  0.2 m/s in
autumn to 2.8  0.3 m/s in spring. Wind speed is important factor
affecting the range and spreading of bioaerosols especially when it
blows >5 m/s [40].
4 Summary and conclusions
In this study the main emission of microorganisms to atmospheric
air was from the mechanical sewage treatment devices of the WWTP
(the grate chamber, the grit chamber, the preliminary settling tank).
The facilities of biological sewage treatment of the plant were
equipped with a fine bubble deep aeration system, which did not
cause any larger turbulences and, consequently, did not generate
large amounts of bioaerosols.
In the air samples obtained on the WWTPs premises, 25 species of
the Enterobacteriaceae including pathogenic bacteria Salmonella spp.,
Klebsiella pneumoniae and potentially pathogenic E. coli were isolated.
At the fence and in the immediate surroundings of the WWTP, only
species belonging to the genera Pantoea were identified. This suggests
that fecal bacteria were mainly dispersed in the area of the WWTP.
Longer survival of Pantoea in the environment may have been
caused by the presence of carotenoid pigments in their cells, which
protected them against solar radiation.
The presence of enteric bacteria, especially Enterobacteriaceae
reflects the level of air pollution with bioaerosols from sewage
and is an important factor in monitoring the quality of the air
around WWTPs.

Variable Temperature Humidity Wind Acknowledgments

(8C) (%) speed
(m/s) This study was supported by Grant No 3 T09D 079 28 by the Ministry
of Science and Higher Education (Poland).
Heterotrophic bacteria (HPC) 0.384 0.207 0.073 The authors would like to thank the manager of WWTP in
Enterobacteriaceae 0.053 0.295 0.175 Ostroda, Malgorzata Tomczykowska, for allowing us to collect air
/
(Endo medium)
Enterobacteriaceae 0.217 0.066 0.206 samples at the WWTP.
(Chromocult medium)
Enterococci 0.203 0.131 0.082 The authors have declared no conflict of interest.
Staphylococci 0.180 0.286 0.168
Actinomycetes 0.321 0.176 0.014
Molds 0.528 0.175 0.117 References
Yeasts 0.204 0.337 0.108
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