Atmospheric Environment 43 (2009) 56985701

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Atmospheric Environment
journal homepage: www.elsevier.com/locate/atmosenv

Short communication

Particle size distribution of airborne Aspergillus fumigatus spores emitted

from compost using membrane ltration
L.J. Deacon a, d, L.J. Pankhurst a, G.H. Drew a, E.T. Hayes b, S. Jackson c, P.J. Longhurst a, J.W.S. Longhurst b,
J. Liu c, S.J.T. Pollard a, S.F. Tyrrel a, *
a
Centre for Resource Management and Efciency, Sustainable Systems Department, School of Applied Sciences, Craneld University, Craneld, Bedfordshire MK43 0AL, UK
b
Air Quality Management Resource Centre, University of the West of England, Bristol, Faculty of Environment and Technology, Coldharbour Lane, Bristol BS16 1QY, UK
c
Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK
d
Environmental Planning & Sustainability Group, Mouchel, No. 1 Waterhouse Sq. 138-142 Holborn, London EC1N 2ST, UK

a r t i c l e i n f o a b s t r a c t

Article history: Information on the particle size distribution of bioaerosols emitted from open air composting operations
Received 13 February 2009 is valuable in evaluating potential health impacts and is a requirement for improved dispersion simu-
Received in revised form lation modelling. The membrane lter method was used to study the particle size distribution of
14 July 2009
Aspergillus fumigatus spores in air 50 m downwind of a green waste compost screening operation at
Accepted 24 July 2009
a commercial facility. The highest concentrations (approximately 8  104 CFU m3) of culturable spores
were found on lters with pore diameters in the range 12 mm which suggests that the majority of spores
Keywords:
are emitted as single cells. The ndings were compared to published data collected using an Andersen
Bioaerosol
Sampling sampler. Results were signicantly correlated (p < 0.01) indicating that the two methods are directly
Filtration comparable across all particles sizes for Aspergillus spores.
Andersen sampler 2009 Elsevier Ltd. All rights reserved.
Aspergillus

1. Introduction
The implementation of the Landll Directive (EC/31/99) has
stimulated the composting industry in Europe. For example, in the
United Kingdom (UK), the amount of waste composted increased
from 0.06 million tonnes in 1994 to an estimated 3.6 million tonnes
in 2007 (Smith and Pocock, 2008). The majority of UK organic waste
(79%) is composted using mechanically turned open-air windrows,
a technology which has limited controls over emissions of parti-
culates to the air (Smith and Pocock, 2008). The release into the air
of microorganisms which originate in the feedstock or which
participate in the composting process, particularly as a result of
activities such as shredding, turning and screening has been well
documented (Lacey, 1997; Reinthaler et al., 1997; Wheeler et al.,
2001; Recer et al., 2001; Taha et al., 2006). Some of the bioaerosols
emitted from compost have the potential to cause harm to humans
if inhaled. The mould Aspergillus fumigatus is an example.
A. fumigatus ourishes within stored organic material, and is
released in elevated quantities during composting operations (in
concentrations exceeding 106 colony forming units (CFU) m3)
* Corresponding author. Tel.: 44 1234 750111.
E-mail address: s.tyrrel@craneld.ac.uk (S.F. Tyrrel).
(Dutkiewicz, 1997; Reinthaler et al., 1997; Swan et al., 2002, 2003;
Taha et al., 2006). Repeat exposure to A. fumigatus is associated with
a range of respiratory conditions, including extrinsic allergic
alveolitis and chronic bronchitis (Dutkiewicz, 1997; Swan et al.,
2003). Although gross exposure to spores (>106 CFU m3) is usually
required to trigger an immune response (Swan et al., 2003),
A. fumigatus is an opportunistic pathogen. Low levels of exposure
may pose an increased risk of invasive aspergillosis in the immu-
nocompromised (Douwes et al., 2003; Swan et al., 2003). Immu-
nological investigations have found increased levels of antibodies
to A. fumigatus amongst compost workers, raising concerns about
the long-term implications of occupational exposure (Bunger et al.,
2000). The health impacts of particulate matter depend partly upon
where they are deposited in the respiratory tract and this in turn is
inuenced by particle size (Park et al., 2009). The size distribution
of bioaerosols emitted from composting facilities is also of interest
to researchers because of the link to dispersion (Taha et al., 2007).
Individual cells are estimated to settle at comparatively low rates
(approximately 3 cm min1) (Swan et al., 2003) which are unlikely
to affect airborne transport off site. Wheeler et al. (2001) postulated
that bioaerosols released during composting may include a range of
particle sizes, due to clumping, which may reduce distance of travel
from source due to preferential deposition of larger particles. No
measurements were made by Wheeler et al. (2001) of particle size

1352-2310/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.atmosenv.2009.07.042
 L.J. Deacon et al. / Atmospheric Environment 43 (2009) 56985701 5699

to substantiate the hypothesis however. The current lack of infor-
mation on the particle size distribution of bioaerosols emitted from
composting limits our ability to model dispersion (Wheeler et al.,
2001; Swan et al., 2003). Although culturable microorganisms
represent only a small fraction of total airborne microorganisms,
cultivation methods are still widely used and remain the principal
available means of generating species-specic information (Parat
et al., 1999). The Andersen 6-stage impactor is widely used to
enumerate airborne culturable microorganisms and to separate
them according to particle size (Andersen, 1958; Reinthaler et al.,
1997; The Composting Association, 1999; Gorny et al., 1999; Tham
and Zuraimi, 2005; Godish and Godish, 2007). Andersen samplers
separate out particles according to their aerodynamic diameter and
impact the different fractions on to agar for viable cell enumeration.
Each stage of the sampler is representative of the human respira-
tory system and the physical barriers that prevent movement of
particles into the lungs (Table 1): stage 1 [nose and mouth], stage 2
[pharynx], stage 3 [trachea and primary bronchi], stage 4
[secondary bronchi], stage 5 [terminal bronchi] and stage 6 [alveoli]
(Andersen, 1958; Reinthaler et al., 1997). Gorny et al. (1999) used
the 6-stage Andersen sampler in a study of size distribution of
bacterial and fungal bioaerosols in indoor air. They concluded that
the majority of Aspergillus spp. In indoor air were present as single
spores. In a study of bioaerosols in ambient outdoor air, Jones and
Cookson (1983) were able to demonstrate using a 2-stage Andersen
sampler that 95% of airborne A. fumigatus were present in the
potentially respirable fraction (<8 mm). As with all bioaerosol
measurement devices, the Andersen sampler has some disadvan-
tages. Specically in the context of composting facilities, it has the
drawback of becoming easily overloaded where bioaerosol
concentrations are high (Reinthaler et al., 1997). There is growing
interest in the use of the membrane-ltration sampling method as
an alternative to the Andersen sampler for capturing bioaerosols in
waste processing environments (Kildes and Nielsen, 1997; Taha
et al., 2006; Adhikhari et al., 2006). German standards have
recently been developed for the sampling of moulds in air using
gelatine and polycarbonate membrane lters (VDI 4252). These
standards have been adopted as the basis for new International
Organisation for Standardisation air quality standards currently
under development (ISO 16000-17). In the context of the wider use
of membrane ltration in bioaerosol studies at composting facilities
and the importance of improving our knowledge of bioaerosol size
characteristics in this environment this paper describes an attempt
to quantify the particle size distribution of A. fumigatus emitted
from a composting facility. This short communication concludes
with a comparison of measured data with previously published
data collected under similar conditions using an Anderson sampler.
waste windrow composting facility in central England, with
a capacity of 25,000 tonnes per annum. The facility is approxi-
mately 500 m from the nearest residence, and approximately
600 m upwind of a village. The samples were collected 50 m
downwind from two screening processes, close (10 m) to the
compost windrows. The wind was predominantly from the S-W,
the site is aligned along an S-S-S-W/N-N-E axis, therefore the
prevailing wind roughly follows the long axis of the site. Medium
ow, personal aerosol lter samplers (SKC Universal dust and
vapour sampling pumps, model SKC PCXR8) operated at a ow rate
of 2.2  0.1 L min1. Pumps were attached to SKC IOM (Institute of
Occupational Medicine) particulate sampling heads, loaded with
IOM multi-dust plastic cassettes, with Tygon tubing and
positioned equidistant along a sampling bar, at a height of 1.7 m
(Taha et al., 2007). Each sample cassette contained one of the six
sizes of polycarbonate lter used (Table 1). Sampling pumps were
run for 30 min. Three sampling runs were completed whilst
compost was being screened to permit assessment of repro-
ducibility. Weather conditions were favourable, with partial cloud
cover, a maximum temperature of 16  C and wind speeds between
3 and 6 m s1. After sampling, lter cassettes were removed from
the sampling heads and placed into a sterile container containing
buffer solution (NaCl 1 g L1 and 3 drops of Tween 80 L1, made up
to 1 L with sterilised ddH2O), agitated to ensure buffer covered the
polycarbonate lter (to avoid cell desiccation) and placed at 4  C for
transport. Once in the laboratory (within 24 h), lters were
removed from the sampling head under aseptic conditions and re-
suspended in the vial by shaking for 2 min. The suspensions were
diluted to a common logarithm order (101 and 102) and aliquots
of 100 mL of each dilution transferred onto the centre of Malt Extract
Agar (MEA) 90 mm single-vented Petri dishes (with 0.1 g L1
Chloramphemicol to suppress bacterial growth). The sample was
spread evenly over the agar using a sterile spreader and once
absorbed, the Petri dish was inverted and incubated at 37  2  C in
the dark for 35 days. Colony forming units (CFUs) were enumer-
ated visually and recorded as CFU m3 of sampled air (Taha et al.,
2005, 2006, 2007).
3. Results and discussion
The lter sizes equivalent to the Andersen sampler stage sizes
(Table 1) were plotted against A. fumigatus colony forming units
100000
90000
80000
70000
CFU m-3

60000
2. Methods
50000
40000
Membrane ltration was used to re-create the particle size
distribution obtained using a 6-stage Andersen sampler. A range 30000
of pore-sizes was chosen that matched closely the range of the 20000
Andersen sampler (Table 1). Sampling was performed at a green 10000
0
Table 1 1 2 3 4 5 6
Cut-off sampling characteristics of the membrane lter and the Andersen 6-stage
samplers. Ranges of stages shown in square brackets.
Equivalent stage number

Stage number Polycarbonate lter size (mm)
1 8.0 [8.0]
2 5.0 [5.08.0]
3 3.0 [3.05.0]
4 2.0 [2.03.0]
5 1.0 [1.02.0]
6 0.65 [0.651.0]
Andersen pore size (mm) Fig. 1. Particle size distribution of Aspergillus fumigatus as captured using membrane
ltration, represented as colony forming units m3 of air, isolated at 50 m downwind of
7.0 [7.0]
screening activities at an industrial scale green waste composting facility. Bars indicate
4.7 [4.77.0]
means (n 3); whiskers show standard error. Equivalent stage numbers indicate
3.3 [3.34.7]
pore-sizes of lters used that are similar to the stage sizes in an Andersen sampler;
2.1 [2.13.3]
stage 1 8.0 mm, 2 5.0 mm, 3 3.0 mm, 4 2.0 mm, 5 1.0 mm and 6 0.65 mm.
1.1 [1.12.1]
(For interpretation of the references to colour in this gure legend, the reader is
0.65 [0.651.1]
referred to the web version of this article.)
5700 L.J. Deacon et al. / Atmospheric Environment 43 (2009) 56985701

(CFU) m3 of air sampled as a particle size distribution (Fig. 1). the facility). For comparison with Reinthaler et al. (1997) the
A. fumigatus concentrations measured at a distance of 50 m particle size distribution from the lter experiment was converted
downwind of the screening operations were in the range of to the same units (percentage of cells at each stage against the total
2  1048  104 CFU m3. Pore size was found to inuence the number recorded) and plotted against the published data (Fig. 2).
recovery of A. fumigatus spores. A steady increase in counts of The relationship between fungi collected by our study and the
A. fumigatus was observed as the lter pore diameters decreased. Reinthaler et al. (1997) work is directly correlated. A p-value of less
The lowest recovery was found on the 8 mm pore diameter lter than 0.01 was recorded demonstrating that lter results and
(stage 1) and peak recovery was found at stages 4 [lter size 2.0 mm, Andersen results are directly comparable across all lter sizes, with
range 2.03.0 mm] and 5 [lter size 1.0 mm, range 1.02.0 mm]. There a percentage route mean squared error of 2.56% maximum varia-
is an approximate 4-fold increase in A. fumigatus CFU m3 from tion between methods. This is a very strong relationship and
stage 1 to stage 5. A. fumigatus conidia are typically 23.5 mm in suggests that the Andersen sampler and membrane lter methods
diameter (Raper and Fennell, 1965; Webster, 1980; Sutton et al., produce comparable results for A. fumigatus spores. This lends
1998). This would place them between equivalent stages 4 and 5 further support to the notion that a proportion of the spores
(Fig. 1). We would therefore expect stages 4, 5 and 6 to have the emitted from compost are in the form of aggregates. The Andersen
highest recoveries if a signicant proportion of the A. fumigatus sampler does not incur lter-related problems such as adhesion, so
spores were present as individual cells. Ongoing work using the we would infer that the spores collected in stages 13 were present
scanning electron microscope (SEM) to visualise spores collected in clumps or attached to other debris emitted from the compost.
from air at composting facilities on lters conrms that a high
proportion of bioaerosols in this environment are present as single
4. Conclusion
cells (data not shown). It seems logical that moulds spores may
have adaptations that minimise the chances of aggregation and
In this study we evaluated the potential for membrane ltration
therefore encourage greater distances of airborne travel.
to be used to assess the size distribution of airborne culturable
Aspergillus spores were also recovered using lters with pore
spores of A. fumigatus at an operational composting facility. We
diameters as large as 8 mm. This suggests that a proportion of the
conclude that the membrane-ltration method is capable of
airborne Aspergillus spores were in the form of clumps or aggre-
producing comparable results to the Andersen sampler for
gates as suggested by Wheeler et al. (2001). Another possibility is
Aspergillus spores. This work suggests that the majority of
that single spores impacting the lter between pores are not always
Aspergillus spores emitted from compost are present as individual
drawn through the pore due to adhesion to the lter surface.
spores, although there is evidence that aggregation also occurs but
Unpublished observations using the SEM provide evidence that
to a lesser extent. This research contributes to an emerging under-
both of these mechanisms occur. We have compared our ndings
standing of bioaerosol size distribution in emissions from com-
with those derived from a very closely related journal paper. Our
posting and in the air as it disperses downwind of the source.
particle size distribution data are in agreement with Reinthaler
Further investigations are needed, combining culture methods and
et al. (1997) who sampled a green waste composting facility for
non-culture dependent methods, such as the optical particle
moulds using an Andersen sampler during sieving (screening)
counter used by Byeon et al. (2008), to study the size distribution of
activities. The data used from the Reinthaler et al. (1997) paper
bioaerosols emitted from composting. Such advancements are
was selected from available datasets from a range of areas of
needed to improve our ability to simulate downwind dispersion and
a composting facility (drop-off, composting areas I & II, sieving
to inform assessments of risk to site workers and the general public.
(screening) and sorting). The sieving (screening) activity data
was selected as it best matched our experimental design
(i.e. immediately downwind from a sieving (or screening) area of Acknowledgement

We acknowledge the funding of this work by the Joint

Andersen sampler (percentage of total moulds recorded by

35 Environment and Human Health Programme of the UK Natural

Environment Research Council (NERC grant NE/E008534/1).
5
30

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