airborne microorganisms emitted from wastewater treatment plant treating domestic wastewater and...

Anna Gotkowska-Płachta1

Zofia Filipkowska1

Ewa Korzeniewska1

Wojciech Janczukowicz2

Beverly Dixon3

Iwona Gołas1

Damian Szwalgin1

1Faculty of Environmental Sciences,

Department of Environmental

Microbiology, University of Warmia

and Mazury in Olsztyn, Olsztyn–

Kortowo, Poland2Faculty of Environmental Sciences

and Fisheries, Department of

Environment Protection Engineering,

University of Warmia and Mazury in

Olsztyn, Olsztyn–Kortowo, Poland3Department of Biological Sciences,

California State University, Hayward,

CA, USA

Research Article

Airborne Microorganisms Emitted from WastewaterTreatment Plant Treating Domestic Wastewater andMeat 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

In recent years the number of wastewater treatment plants (WWTPs)

has increased worldwide. As a rule, such facilities are located outside

urban settlements, but as towns grow, WWTPs are increasingly

found very close to urbanized areas [1]. Pollutants emitted during

the process of sewage and wastewater treatment (odors, bioaerosols,

chemicals) can be harmful to human and animal life [2–5]. Biological

aerosols produced during wastewater treatment (discharging,

mixing, aerating, and spraying of sewage) can contain various

pathogenic microbes, such as viruses, fungi and bacteria, especially

intestinal bacteria from the Enterobacteriaceae [6–11]. Some of

these microorganisms are associated with toxic pneumonia

(inhalation fever, organic dust toxic syndrome ODTS), chronic

bronchitis, asthma, and other disorders [2, 3, 8, 11–14]. The dispersal

of bioaerosols outside the facilities of WWTPs, as well as the

type, amount and survival rate of airborne microorganisms depend

on physicochemical and meteorological air conditions, landscape

features, the time of the day, the season, and the type of treatment

technology [9, 15–21]. Aerosols containing pathogenic micro-

organisms generated by the technological facilities at WWTPs can

be dispersed over considerable distances, producing adverse effects

on living organisms, primarily by inhalation.

The main objective of this study was to analyze the bacterial

(especially from Enterobacteriaceae) and fungal contamination of

atmospheric air on the premises and in the surroundings of the

WWTP, which treated predominantly domestic sewage and waste-

water from the meat processing industry. The presence of these

microorganisms and their identification reflect the level of air

pollution with bioaerosols from sewage, and are a vital consider-

ation for monitoring the air quality around WWTPs. Wastewater

treatment using activated sludge and fine bubble aeration

utilized at this WWTP, is one of the most commonly used techno-

logies [16, 19, 20, 22]. Therefore, the results may serve as a good

comparison with other sites using the same type of technology.

2 Materials and methods

2.1 Area of study

The wastewater treatment plant in Ostroda in the north-east

of Poland was refurbished in 2001–2002. 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

Correspondence: Dr. A. Gotkowska-Pl

/

achta, Faculty of EnvironmentalSciences, Department of Environmental Microbiology, University ofWarmia and Mazury in Olsztyn, R. Prawochenskiego Street 1, 10–957Olsztyn–Kortowo, PolandE-mail: [email protected]

Abbreviations: CFU, colony forming unit; HPC, heterotrophic bacteria;WWTP, wastewater treatment plant

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2013, 41 (5), 429–436

429

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 WWTP’s area there were

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

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� 10�2

(in spring, summer, autumn) and 3� 10�2 m3 (in winter) for HPC and

fungi. For other groups of bacteria was 6� 10�2 (in spring, summer,

autumn) and 10� 10�2 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 3–5 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

Figure 1. Technological scheme of the WWTP and the � sampling sitelocation: � C: control site; Mechanical treatment (1: grate chamber, 2: gritchamber, 3: retention chamber, 4: preliminary settling tank); Biologicaltreatment (5: pre-denitrification tank, 6: nitrification and denitrificationtanks, 7: secondary sedimentation tank); Surrounding (8: fence, 9: 50 m,10: 100 m, 11: 200 m from the fence); A: sludge field.

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)of media used in

microbiological analyses

Temperature ofincubation (8C)

Time ofincubation

Timea) of agardishes exposure

(min) (sedimentationmethod)

Volume(�10�2 m3) ofair samplesb)

(impact method)

Heterotrophic bacteria (HPC) Nutrient agarc) 26 72 h 10 2–3Enterobacteriaceae Chromocult coliform agarc) 37 24 h 30 6–10

Endo agarc) 37 24 hEnterococci Membrane-filter enterococcus selective

agar acc. to Slanetz and Bartleyc)37 72 h 30 6–10

Staphylococci Chapman agarc) 37 48 h 30 6–10Pseudomonas fluorescens King agar Bc) 26 48 h 30 6–10Actinomycetes Pochon mediuma) 26 7 days 30 6–10Fungi (molds, yeastsand yeast-like fungi)

RBC medium (rose-bengalchloramphenicol agar) c)

25� 3 3–7 days 10 2–3

a) References: Polish standards [24].b) Depends on season: in spring, summer, autumn: 2–6; in winter 3–10.c) Merck Poland.

430 A. Gotkowska-Płachta et al.


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 Chapman’s 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 Spearman’s 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 WWTP’s premises and

surroundings, during the whole time of the study

Variable Control site n¼ 24 Mechanical treatmentb) n¼ 96 Biological treatmentc) n¼ 72 Surroundingsd) n¼ 96

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

Heterotrophic bacteria

(HPC)

8.9� 102 5.9� 101/1.1� 103 8.8� 102 0.8� 101/1.2� 104 3.5� 102 0.8� 101/4.8� 103 2.9� 102 0.8� 101/6.5� 103

Enterobacteriaceae

(Endo medium)

0 0/0 0.3� 101 0/1.5� 103 0 0/2.9� 101 0 0/3.6� 101

Enterobacteriaceae

(Chromocult medium)

0 0/1.1� 102 7.6� 101 0/2.3� 103 1.9� 101 0/8.0� 102 0 0/2.1� 102

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 inthe whole period of the study.

Airborne Microorganisms Emitted from Wastewater Treatment Plant 431


3.3 Quantitative and qualitative composition of

microorganisms in the air samples collected from

the area and surroundings of the WWTP

In the area and the surroundings of the WWTP 2016 air samples were

analyzed. The counts of HPC found in the air samples in the WWTP’s

area were between 0.8� 101 and 1.2� 104 CFU/m3. In the mechanical

sewage treatment devices (the grate chamber and the grit chamber)

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),

but higher than those collected near the facilities of biological

sewage treatment of the plant (3.5� 102 CFU/m3) and its surround-

ings (2.9� 102 CFU/m3; Tab. 2).

The bacterial counts of fecal bacteria from Enterobacteriaceae

ranged from 0 to 2.3� 103 CFU/m3 in the samples of air obtained

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

mechanical sewage treatment devices (the grate chamber and the

grit chamber) of the plant could have been caused by the constant

flow of raw wastewater through the aerated grit chamber, in which

small droplets of bioaerosols were produced and dispersed to the air

by wind. Another factor which may have raised the microbial counts

at these sampling sites, was a worm wheel conveyor, periodically

switched on to carry sand from the grit chamber to a nearby con-

tainer. At these times, even a light wind could have dispersed large

quantities of microorganisms.

The level and range of bioaerosols emitted to the air from WWTPs

depends on the type of technology employed for wastewater treat-

ment and aeration system [15–17, 20–22, 27]. Microorganism emis-

sion is lower when depth aeration, rather than surface, is employed.

In the examined sewage treatment plant the aeration chamber was

equipped with a fine bubble deep aeration system which did not

produce large turbulence, and consequently did not generate a large

amount of bioaerosols. These results correspond with the results of

other authors conducting research in similar operational conditions

[9, 20, 28, 29]. When surface aeration was employed, the emission of

microorganisms in the areas adjacent to aeration chambers was the

highest [15, 20].

In the air samples obtained on the premises of the WWTP,

165 identified strains belonging to 25 bacteria species from

Enterobacteriaceae were isolated. At the fence and in the immediate

surroundings of the plant up to 200 m, only the species belonging

to the genus Pantoea were identified (Tab. 3). The Pantoea bacteria

are widely distributed in nature and isolated from numerous eco-

logical niches, including plants, water, soil, humans, and animals.

The presence of Pantoea bacteria in the surroundings of the WWTP

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

Site Bacteria identified

Control site WWTP area Pantoea spp. 3a)

Grate chamber Enterobacter sakazakiiGrit 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. 2Secondary sedimentation tank Pantoea spp. 2SurroundingsFence Pantoea spp. 350 m Pantoea spp. 2100 m Pantoea spp. 3200 m Pantoea spp. 2

a) 1, 2, 3: different biotypes within a species.

Table 4. The occurrence of molds in studied air samples in the WWTP’s

area and its surrounding

Genus of molds WWTP area WWTP surrounding

Absidia 1, 2 9Actinomucor C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10Alternaria C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11Aspergillus C, 1, 2, 3, 4, 5, 6 8, 9, 10, 11Botrytis 7 11Chaetomium C, 5Chrysosporium C, 2, 3, 5, 7 8, 9, 10, 11Cladosporium C, 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11Cunninghamella C, 6Diplosporium C, 4Doratomyces 9Fusarium C, 5 8Geotrichum C, 1, 2, 3, 5, 6, 7 8, 9, 10Gliocladium 8Mucor C, 1, 2, 3, 4, 5, 6, 7 8, 9, 11Nigrospora 6Penicillium C, 1, 2, 5, 7 8, 10Phoma CRhizopus C, 1, 3 8Scopulariopsis C, 2, 3, 4, 6 8Sporothrix CThamnidium C, 2, 3Trichoderma C, 4, 5 8Trichothecium C, 4, 5, 6 10

C: control site, WWTP’s area (1: grate chamber, 2: grit chamber, 3:retention chamber, 4: preliminary settling tank, 5: predenitrifica-tion tank, 6: nitrification and denitrification tanks, 7: secondarysedimentation tank), surroundings (8: fence, 9: 50 m, 10: 100 m,11: 200 m).



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 WWTP’s 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 WWTP’s 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 WWTP’s premises

(median¼ 1.4� 101), in the surroundings (median¼ 1.0� 101), and

at the control sites (median¼ 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 WWTP’s premises the most

abundant yeasts were Candida and Cryptococcus, in the WWTP’s

immediate surroundings Candida and Rhodotorula were isolated

sporadically.

The air samples of WWTP’s 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].

Soil and plants are probably the main source of molds in the

air of WWTP’s surroundings. Yeasts occurred sporadically in air

samples from the WWTP’s 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 WTTP’s 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 (p¼ 0.0054);

Enterobacteriaceae (on Chromocult medium p¼ 0.038, and on Endo

medium p¼ 0.017); actinomycetes (p¼ 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 1.1–2.1 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).



Figure 2. Average numbers (CFU/m3) of (A) het-erotrophic bacteria (HPC), (B) Actinomycetes,(C) Enterobacteriaceae on Chromocult medium,(D) Enterobacteriaceae on Endo medium, fromair samples collected by different methods (S,sedimentation; I, impact). Independent variable(assembling): method. RMS� random meansquare; N, number of samples; p, significancelevel.

Figure 3. Average numbers (CFU/m3) of (A) het-erotrophic bacteria (HPC), (B) Enterobacteriaceaeon Chromocult, (C) Staphylococci, (D)Actinomycetes, (E) Molds, (F) Yeasts, from airsamples collected at the different sites duringthe whole season of study. Independent variable(assembling): season. RMS� random meansquare; N, number of samples; p, significancelevel.



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 (r¼�0.286, p¼ 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 (r¼ 0.384, p¼ 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 (r¼ 0.321, p¼ 0.002) and for molds (r¼ 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.

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

(r¼ 0.217, p¼ 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 WWTP’s 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.

Acknowledgments

This study was supported by Grant No 3 T09D 079 28 by the Ministry

of Science and Higher Education (Poland).

The authors would like to thank the manager of WWTP in

Ostroda, Mal/gorzata Tomczykowska, for allowing us to collect air

samples at the WWTP.

The authors have declared no conflict of interest.

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Variable Temperature(8C)

Humidity(%)

Windspeed(m/s)

Heterotrophic bacteria (HPC) 0.384�� 0.207 �0.073Enterobacteriaceae(Endo medium)

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