Journal of Applied Microbiology 2000, 88, 845ÿ852

Evaluation of the environmental impact of microbial aerosols

generated by wastewater treatment plants utilizing different
aeration systems
G. Brandi, M. Sisti and G. Amagliani
Institute of Toxicologic, Hygienic and Environmental Science, University of Urbino, Urbino, Italy

18/10/99: received 20 October 1999, revised 23 December 1999 and accepted 4 January 2000

Using three sampler devices (SAS, Andersen

G . B R A N D I , M . S I S T I A N D G . A M A G L I A N I . 2000.
Six-Stages and All Glass Impinger), the environmental impact of bacterial and fungal
aerosols generated by municipal wastewater treatment plants operating with different
methods of sludge oxygenation were evaluated. The highest microbial concentrations were
recovered above the tanks (2247 cfu mÿ3) and in downwind positions (1425 cfu mÿ3),
where a linear correlation (P < 0
05) was found between the quantity of sewage treated and
the entities of microbial aerosol dispersion. Moreover, an exponential increase (P < 0
05) in
the bacteria recovered from the air occurred at increasing times of treatment. However,
after long-term plant operation, high bacterial and fungal concentrations were found in
almost all of the sites around the plant. Coliforms, enterococci, Escherichia coli and
staphylococci were almost always recovered in downwind positions. Considerable fractions
(20±40%) of sampled bacteria were able to penetrate the ®nal stages of the Andersen
apparatus and thus, are likely to be able to penetrate the lungs. The plant operating with a
®ne bubble diffused air system instead was found to generate rather low concentrations of
bacteria and fungi; moreover, staphylococci and indicator micro-organisms were almost
absent. Finally, salmonellae, Shigella, Pseudomonas aeruginosa and Aeromonas spp. were not
detected in either of the plants. The results indicate a remarkable dispersion of airborne
bacteria and fungi from tanks in which oxygen is supplied via a mechanical agitation of
sludge, and suggest the need to convert them to diffused aeration systems which pose a
lesser hazard for human health.

INTRODUCTION hazardous for exposed subjects, ®rstly for plant workers as

a consequence of direct contact with the contaminated
Aerosols containing micro-organisms may be generated
materials/substrates or by inhalation of aerosolized micro-
through natural processes and by human activity. It has
organisms. Moreover, a potential risk for visitors to the
been reported that aerosols are capable of transporting
plants and for the surrounding population cannot be
micro-organisms over long distances (Bovallius et al. 1978)
excluded. Various potentially infectious agents have fre-
and, depending on the source, that they may be able to
quently been recovered from the air around wastewater
produce infections (Fraser 1980; Sattar and Ijaz 1987),
plants (Hickey and Parker 1975; Millner et al. 1980;
asthmatic problems (Gravesen 1979) and other health
Fannin et al. 1985) and a high prevalence of antibodies
effects (Jacobs 1989; Burrel 1990) in susceptible subjects.
against several enteric viruses has been found in exposed
The aeration tanks of wastewater treatment plants are
subjects (Clark 1981a; Heng et al. 1994), although this is
recognized as important sources of microbial aerosols
not necessarily related to aerosols. Clear evidence of the
(Napolitano and Rowe 1966; Kenline and Scarpino 1972;
adverse health effects caused by exposure to micro-organ-
Cannon 1983; Fannin et al. 1985) and therefore might be
ism-containing aerosols, however, has not yet been pre-
sented in a satisfactory manner (Clark et al. 1981b; Stener
Correspondence to: G. Brandi, Istituto di Scienze Tossicologiche Igienistiche e
1986; Maguire 1993), and as far as is known, outbreaks due
Ambientali, UniversitaÁ di Urbino, Via S. Chiara, 27, 61029 Urbino (PU) to aerosols generated during the wastewater treatment pro-
Italy (e-mail: brandi@bio.uniurb.it). cess have never been reported. Furthermore, the health
= 2000 The Society for Applied Microbiology
846 G . B R A N D I E T A L .
risks for populations living near wastewater treatment
plants remain poorly investigated. The aim of the present
study was to obtain more information about the environ-
mental microbial dispersion from wastewater treatment
plants by evaluating the densities and types of airborne
bacteria and fungi recovered in the vicinity of the aerated
tanks. For this purpose, two different plants were selected.
The ®rst is operative only in the summer and uses a
mechanical agitation for the aeration of the settled sewage.
The other is operative year-round and the oxygen for sew-
age aeration is supplied via a ®ne bubble diffused air sys-
tem.
MATERIALS AND METHODS
Wastewater treatment plants
Two wastewater treatment plants located along the north-
west Adriatic coast were studied. The ®rst (hereafter
referred to as Plant A) is operative only in the summer and
receives 7500 mÿ3 dÿ1 of wastewater during its peak of
activity, equivalent to about 30 000 inhabitants. This plant
uses a mechanical aeration of settled sewage (activated
sludge process). The second plant (Plant B) operates con-
tinuously throughout the year and treats 15 500 mÿ3 dÿ1 of
sewage in winter, 16 000 mÿ3 dÿ1 in spring and 22 000 mÿ3
dÿ1 in summer, corresponding to the equivalent of about
62 000, 64 000 and 88 000 inhabitants served, respectively.
The oxygen in the tanks is supplied by a ®ne bubble dif-
fused air system.
The sampling of aerosols was carried out near the waste-
water aeration tanks as follows: in summer at different peri-
ods before and after start-up of the process in Plant A; and
in winter, spring and summer in Plant B.
Air samplers and micro-organisms assayed
Three different samplers were employed for the detection
and enumeration of aerosolized micro-organisms. The SAS
(Surface Air System) impactor aspirates air at a ®xed speed
(180 l minÿ1) for a variable time onto a 55 mm contact
plate ®lled with appropriate agar medium. All aerosol sam-
ples were collected after determination of the wind direc-
tion; the sites were at a distance of 2 m upwind, 2, 10
(Plant B) and 20 (Plant A) m downwind, and 2 m laterally,
with respect to wind direction. The sampling time used
varied from 30 s to 2 min, depending on the supposed
microbial concentration in the air. The micro-organisms
sampled were total bacteria and fungi, total coliforms,
enterococci, Escherichia coli and staphylococci.
The Andersen Six-Stage Viable Particle Sampler (here-
after Andersen) contained six Petri dishes ®lled with an
appropriate agar medium. Each stage has 400 holes with
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852
diameters that range from 1
81 mm in the ®rst stage to
0
25 mm in the sixth stage. Aerosol samples were collected
1
5 m above and 2 m downwind of the aeration tanks. The
sampling time was 20 min, with a constant sampling ¯ow
rate of 28
5 l minÿ1. The sampler was used to evaluate the
total bacterial count.
The All Glass Millipore Impinger (hereafter Impinger)
containing appropriate enrichment broth medium was posi-
tioned 2 m downwind from the tanks. The sampling time
was 30 min with a ¯ow ratio of 12
5 l minÿ1. This sampler
was used to collect salmonellae, Shigella, Aeromonas spp.
and Pseudomonas aeruginosa.
All collected samples were transported to the laboratory
within 3±4 h of sampling and were incubated at 37  C for
bacterial counts, at 20  C for fungal counts and at 45  C for
E. coli counts. The results are expressed as the number of
colony-forming units per cubic metre (cfu mÿ3) of air.
At the same time as aerosol sampling, the temperature
and relative humidity were monitored, using an aspirated
psychrometer, and wind speed, by an anemometer.
Culture media
The solid culture media were tryptic glucose yeast agar
(Oxoid) for total bacterial counts, Sabouraud dextrose agar
(Oxoid) for total fungal counts, violet red bile lactose agar
(Oxoid) for total coliforms and E. coli, Bacto m. enterococ-
cus agar (Difco) for enterococci, mannitol salt agar (Oxoid)
for staphylococci (colonies grown in this agar were pro-
cessed for the coagulase test using the Staphytect plus,
Oxoid), SS agar modi®ed (Oxoid) for salmonellae and
Shigella, m-Aeromonas selective agar (Biolife) for
Aeromonas, and pseudomonas CN medium (Oxoid) for Ps.
aeruginosa. The liquid enrichment media used for Impinger
were selenite cystine broth (Oxoid) for salmonellae and
Shigella, nutrient broth (Oxoid) for Ps. aeruginosa and alka-
line peptonate water for Aeromonas.
Statistical analyses
A linear and exponential regression (both evaluated by
ANOVA) and a Spearman rank correlation were used.
RESULTS
The results on airborne bacteria and fungi recovered from
Plant A are shown in Fig. 1. Before the plant was started
up (Period A), the concentrations of total bacteria and
fungi was found to be very low in all ®ve positions selected.
Enterococci were not detected while coliforms and staphy-
lococci were only rarely recovered during sampling. After
plant operation commenced (Periods B±E), a progressive
 MICROBIAL AEROSOL FROM AERATION TANKS 847

(a) (b)
640 1250

1000
480

750
cfu m–3

cfu m–3
320
500

160
250

0 0
A B C D E A B C D E
Periods Periods
(c) (d)
200 40

150 30
cfu m–3

cfu m–3

100 20

50 10

0 0
A B C D E A B C D E
Periods Periods
(e) (f)
60 60

45 45
cfu m–3

cfu m–3

30 30

15 15

0 0
A B C D E A B C D E
Periods Periods

Fig. 1 Total bacterial and fungal counts, coliforms, enterococci, Escherichia coli and staphylococci recovered from Plant A. All the values
represent the mean of three separate air samplings performed with the SAS instrument and are expressed as cfu mÿ3. Positions:
1 (&) ˆ2 m upwind, 2 (Q) and 3 (R) ˆ 2 m, respectively, on the right and on the left with respect to the wind direction, 4 (W) and
5 ( & ) ˆ 2 and 20 m downwind, respectively. Periods: before (A) and 1 (B), 3 (C), 12 (D) and 25 (E) days after plant activity was started.
(a) Total bacterial count; (b) total fungal count; (c) total coliforms; (d) enterococci; (e) Escherichia coli; (f) staphylococci

increase in the presence of aerosolized micro-organisms (P < 0
05) between the total viable bacterial and fungal
was found. The highest concentrations were recovered at counts obtained and the corresponding quantity of sewage
the sites 2 m (Position 4) and 20 m (Position 5) downwind. being treated (Fig. 2). From analysis of panels (a) and (b)
Furthermore, analyses of data obtained in the downwind in Fig. 1, it can be seen that the greatest concentrations of
positions showed a statistically signi®cant correlation bacteria and fungi were found about 4 weeks after the start
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852
848 G . B R A N D I E T A L .

(a) (b)
600
600

500 500

400 400
R = 0·91233 R = 0·98155
P = 0·03075 P = 0·003
cfu m–3

cfu m–3
300 300

200 200

100 100

0 0

0 2000 4000 6000 8000 0 2000 4000 6000 8000

–3 –1 –3 –1
Sewage treated (m d ) Sewage treated (m d )

(c) (d)
1200 1200

1000
1000

R = 0·89822 800 R = 0·96533

800 P = 0·03838 P = 0·00771
cfu m–3

cfu m–3

600
600

400

200

200

200

0
0 2000 4000 6000 8000 0 2000 4000 6000 8000
–3 –1
Sewage treated (m d ) Sewage treated (m–3 d–1)

Fig. 2 Correlation between the concentrations of airborne bacteria [(a) and (b)] and fungi [(c) and (d)] and the amount of sewage treated.
Air was sampled with the SAS at 2 m [(a) and (c)] or 20 m downwind [(b) (d)] of Plant A
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852

of the process (Period E), corresponding to maximum
activity of the plant (7500 mÿ3 dÿ1 sewage treated). Total
bacterial counts ranged from 444 cfu mÿ3 laterally with
respect to the tanks, to 560 cfu mÿ3 downwind, while fun-
gal concentrations ranged from 660 to 1110 cfu mÿ3 in the
same positions. It is interesting to note that with increasing
time from the start-up of activity, microbial aerosol disper-
sion appeared to become more uniformly distributed
around the tanks. Total coliforms and enterococci showed
a similar pattern, although to a different degree, of total
bacterial and fungi aerodispersion, while E. coli was recov-
ered only in Periods C±E. Staphylococci were constantly
recovered at the downwind positions in concentrations that
increased, as it did for the other species of bacteria, with
the time that the plant had been running; 16±40% of colo-
nies were identi®ed as staphylococci coagulase ‡ and there-
fore were probably Staph. aureus. Finally, temperature
(ranging from 24 to 27  C), humidity (from 53 to 77%) and
wind speed (from 1
5 to 4 m sÿ1), were shown to be quite
similar during the different periods of sampling.
A second set of experiments was performed with the
Andersen apparatus above the platforms and at 2 m down-
wind from the tanks of Plant A. As shown in Table 1, the
concentration of total bacteria in the aerosols was already
high, especially on the platforms, just a few days after the
process had started. Using an exponential regression ‡
ANOVA test, it was observed that there was an exponential
increase in the cfu mÿ3 with time, both above the tank (P
ˆ 0
019) and 2 m downwind (P ˆ 0
01). When the same
test was applied taking into account the quantities of sew-
age treated rather than time, the results were not signi®cant
(P > 0
05). The same result was also obtained with the
SAS data (P ˆ 0
009) in the 2 m downwind position. This
would suggest the importance of time in determining the
microbial aerosol concentration. Comparing the data
obtained with SAS (Fig. 1) with those obtained with the
Table 1 Total bacterial counts obtained sampling with the six-stage Andersen device above and 2 m downwind from Plant A before and
after the start-up of plant activity*
Above the tank
Six stages 5th ‡ 6th stage
Period n n
Before 91 35
After 1 day 817 172
After 3 days 1489 494
After 12 days 2247 635
After 25 days 1623 329
*Results are expressed as cfu mÿ3 and represent the mean of three experiments that agreed within 15% of the reported values.
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852
MICROBIAL AEROSOL FROM AERATION TANKS 849
Andersen sampler (Table 1) at the same time at 2 m down-
wind, it is of note that the bacterial counts were higher by
Andersen sampling, suggesting that this device is more ef®-
cient in collecting viable bacteria. As shown in Table 1, the
percentage of bacteria able to reach the 5th and 6th stages
of the Andersen apparatus ranged from about 20 to 40% of
the total bacteria sampled, and these percentages were not
correlated (P ˆ 0
39, Spearman rank correlation test) with
the amount of total bacteria sampled.
All the sampling performed with the Impinger device in
Plant A gave negative results for isolation of salmonellae,
Shigella, Ps. aeruginosa and Aeromonas spp. Sampling (250 l
of air each time) was repeated three times for each period
at 2 m from the tanks in the downwind position.
In Plant B, which uses a ®ne bubble diffused air acti-
vated sludge process and is continuously operative, the
aerosols were sampled with SAS at 2 m upwind, and 2 m
and 10 m downwind, in winter, spring and summer. As
shown in Table 2, the concentrations of both total bacteria
and fungi, and of the various bacterial species studied,
were markedly lower or absent compared with those recov-
ered in the same positions at Plant A. As for Plant A, the
highest concentrations of bacteria and fungi were collected
at the site 2 m downwind from the tanks, i.e., 298 and 222
cfu mÿ3 bacteria, and 147 and 190 cfu mÿ3 fungi, in winter
and summer, respectively. Escherichia coli, total coliforms
and enterococci were not detected, with the exception of
coliforms found 10 m downwind in winter, and one occa-
sion when enterococci were found 2 m downwind in spring.
Staphylococci were only recovered downwind and in low
numbers. The lower airborne bacterial concentrations near
Plant B were also con®rmed using the Andersen sampler
(not shown).
As in the case of Plant A, salmonellae, Shigella, Ps. aeru-
ginosa and Aeromonas spp. were not recovered with the
Impinger.
2 m downwind
Six stages 5th ‡ 6th stage
% n n %
38
4 85 35 41
1
21
0 374 155 41
4
33
1 657 155 23
6
28
2 1425 405 28
4
20
2 1081 378 34
9
850 G . B R A N D I E T A L .

Table 2 Mean micro-organisms concentrations recovered with the SAS in different seasons and sampling sites in Plant B*

Seasons

Micro-organisms Positions Winter Spring Summer

Total bacterial count 2 m upwind 8
9 5
5 66
6

2 m downwind 298
8 11
1 222
10 m downwind 170
5 11
1 105
4
Total fungal count 2 m upwind 27
5 38
8 92
2 m downwind 147
2 38
8 190
10 m downwind 62
3 38
8 106
Total coliforms 2 m upwind nd nd nd
2 m downwind nd nd nd
10 m downwind 1
3{ nd nd
Enterococci 2 m upwind nd nd nd
2 m downwind nd 2
7{ nd
10 m downwind nd nd nd
Escherichia coli 2 m upwind nd nd nd
2 m downwind nd nd nd
10 m downwind nd nd nd
Staphylococci 2 m upwind nd nd nd
2 m downwind 12
4 20
8 24
9
10 m downwind 6
9 8
3 11
1

*Results are expressed as cfu mÿ3 and represent the mean of three experiments that agreed within 15% of the reported values.
{Value of a single sampling.

Comparing the data obtained in Plant A with those for
Plant B, it is clear that higher concentrations of aerosols
containing micro-organisms were generated from the tanks
of the ®rst plant, probably as a consequence of the mechan-
ical aeration of the sludge. To test this hypothesis, the data
obtained for Plant B in this study were compared with
those obtained for the same plant in an earlier study
(Brandi et al. 1993) when mechanical oxygenation was used
for the digestion process. As shown in Table 3, total bacter-
ial and fungal counts, coliforms and staphylococci were
recovered to a much greater extent at the same positions
and seasons when the sludge was mechanically aerated.
DISCUSSION
Airborne micro-organisms generated from wastewater
treatment plants may be a potential source of a wide variety
of health hazards (Hickey and Parker 1975). Therefore, to
ensure the health of plant workers and local residents, it is
also necessary to evaluate the presence and concentrations
of airborne micro-organisms in the proximity of the plants.
With this aim, a study was conducted to evaluate the dif-
ferent factors in¯uencing the microbial concentrations of
aerosols liberated from the aeration tanks. In order to
determine the importance of the oxygenation systems used,
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852
one plant was selected that uses mechanical agitation for
aeration of the sludge and another in which the oxygen was
supplied with a ®ne bubble diffused air system.
Furthermore, as the former plant was operative only in the
summer, serving a mainly tourist population, it was possi-
ble to distinguish the type and quantity of microbial pollu-
tion due to the wastewater treatment from the microbial
background of the air in the speci®c area.
From the data reported in this paper it is clear that the
type of aeration used in the activated sludge process mark-
edly in¯uences the microbial content of the air around the
plant. Mechanical agitation of the sludge generates aerosols
with high concentrations of bacteria and fungi. It is inter-
esting to note that when the plant reached its peak of activ-
ity, high concentrations of airborne bacteria and fungi were
seen as far as 20 m downwind of the tank and 2 m laterally
with respect to the wind direction. This would suggest that
when the digestion process is fully operative, a potential
risk for human health may exist at almost all the sites
around the plant. Consistent with this supposition was the
presence of coagulase positive staphylococci and indicator
micro-organisms (coliforms, E. coli, enterococci) found
above all in the downwind positions.
From a comparison of data obtained with the different
samplers, it is evident that the Andersen was more ef®cient
 MICROBIAL AEROSOL FROM AERATION TANKS 851

Table 3 In¯uence of the sludge oxygenation system on the dispersion of microbial aerosols from Plant B*

Season

Spring Summer

2m 10 m 2m 10 m

Mech{ Bubbl{ Mech Bubbl Mech Bubbl Mech Bubbl

Total bacterial count 483
3 11
1 274
9 11
1 1816
6 222
0 1383
3 105
4
Total fungal count 541
6 38
8 816
6 38
8 2900
0 190
0 5000
0 106
0
Staphylococci 25
9 20
8 25
0 8
3 100.0 24
9 183
3 11
1
Total coliforms 75
2 0 37
2 0 966
6 0 366
6 0
Escherichia coli 0 0 0 0 54
1 0 16
6 0

*Samplings were performed with SAS at downwind positions and are expressed as cfu mÿ3. The values referring to Bubbl. were obtained
in a previous study (Brandi et al. 1993) and represent the mean of three experiments for each season and sampling site that agreed within
15%.
{Aeration of the sludge: Mech ˆ mechanical agitation, Bubbl ˆ ®ne bubble diffused air.

than the SAS in collecting bacteria from the air. This dis-
crepancy was not unexpected as both ®eld and laboratory
studies have frequently reported considerable differences
among the density of collected micro-organisms measured
with different sampling devices (Jensen et al. 1992).
Furthermore, several factors can contribute to underesti-
mation of the bacterial concentration in aerosols. First of
all, underestimation due to a colony masking effect (colony
overlap) should be considered (Chang et al. 1994).
Furthermore, during sampling, microbial damage may
occur due to the impact of the cells on the agar, affecting
the viability and culturability of collected micro-organisms
(Stewart et al. 1995). It is reasonable to assume that the
degree of microbial injury will increase with air¯ow speed.
This would explain, at least in part, the lower cfu counts
obtained here with SAS, which samples airspeeds 6
3-fold
higher than with the Andersen.
The aim of using the Andersen sampler, however, was
also to obtain information on cell density and particle size
(Andersen 1958). As this sampling device sizes the airborne
particles by collecting them in different stages, it is possible
to measure the fraction of respirable particles with the
potential to reach the alveoli. It was observed that this frac-
tion was already greater than 20% (median 30
7) of the col-
lected bacteria. However, the percentage of bacteria able to
penetrate the lungs was not correlated with the degree of
aerosol contamination. This would suggest that the risks
associated with exposure to aerosols could be closely related
not only to the density but also to the type of aerosolized
particles.
= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 88, 845ÿ852
In view of the problem of bacterial damage due to agar
impact and/or cellular dessication on the agar surface as a
result of long exposure to air¯ow, it is thought that neither
the Andersen nor the SAS devices can be used ef®ciently
for sampling bacterial species, such as pathogenic bacteria,
that may be present in very low concentrations in aerosols.
Therefore, to overcome this limitation, a bioaerosol sampler
(Impinger) was selected which uses a liquid medium for
micro-organism collection, although the device was still
unable to recover salmonellae, Shigella, Aeromonas spp. and
Ps. Aeruginosa.
An increasing number of reports have recognized the
presence of bacteria, including various human enteric
pathogens (e.g. Salmonella enteritidis, Shigella, enterotoxic
E. coli, Vibrio cholerae) from several substrates in a viable
but non-culturable state (Roszak et al. 1984; Islam et al.
1993; Cappelier et al. 1999). The presence of viable but
non-culturable pathogenic bacteria in aerosols should not
be excluded.
In conclusion, wastewater treatment plants operating
with mechanical aeration have an environmental impact
that is clearly unfavourable, due to high dispersion around
the tanks of aerosols containing micro-organisms. On the
contrary, aerobic digestion with a submerged microbubble
system seems to pose little risk from airborne transmission
of pathogenic bacteria and fungi to waste-treatment work-
ers and local residents. For this reason, the conversion of
mechanical oxygenation systems to submerged oxygenation
systems of the sludge should be considered.
Although it is dif®cult to evaluate the precise contribu-
tion of microbial aerosols generated from tanks to human
852 G . B R A N D I E T A L .
illness, determination of the micro-organism content and
presence of pathogenic species in air at the wastewater
treatment plant site may provide an insight into their
potential adverse health effects.
ACKNOWLEDGEMENTS
The authors thank M. Rocchi for his support in perform-
ing the statistical analyses. This research was supported by
Italian Ministry of University and Scienti®c and
Technological Research.
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