Development of Techniques to Measure Bioaerosol
Deposition
Michael Jahne
Dr. Shane Rogers, Mentor
Department of Civil and Environmental Engineering
Background and Introduction
Bioaerosols, particles of biological origin suspended in the air, include bacteria, fungi, spores,
pollens, and a host of other whole cells and cell fragments (USEPA, 2004). Often these particles are a
human health risk, associated with asthma and respiratory illness, infections of the lungs and sinuses, and
gastrointestinal disease (Macher et al., 1999). Thus, they present an occupational concern for workers at
bioaerosol sources such as wastewater treatment plants and farming operations, as well as a public health
issue for nearby residents (Johnson et al., 1979; Merchant et al., 2002). Furthermore, there may be
potential for significant migration of suspended particles away from the source, followed by deposition
into surface waters and onto diverse land-use areas.
Despite these concerns, there remain few options for outdoor bioaerosol monitoring, especially
methods for direct measurement of their deposition (Cox and Wathes, 1995.) Current deposition
measurement techniques involve collection onto a semi-solid agar surrogate surface, whose colonies are
counted following incubation. However, this method has important limitations that reduce its
applicability, including the potential for overlapping colonies, many organisms’ inability to grow on agar
plates, and the time delay required before identification and quantification (Chang et al., 1994; Colwell
and Grimes, 2000). To address these issues, alternative analysis techniques that do not require culturing
have been developed, including the use of fluorescent microscopy to enumerate collected cells and
application of real-time polymerase chain reaction (PCR) to air samples for rapid identification and
quantification of organisms (Terzieva et al., 1996; An et al., 2006). To improve actual deposition sample
collection, a static water surface sampler with an aerodynamically designed surrogate surface has been
developed at Clarkson University, and was recently applied in the analysis of bioaerosol deposition by
Sahu, et al. (2005).
The objective of the current study is to build upon previous work by combining these novel
techniques for deposition sample collection with advanced microbial analysis, using both fluorescent
microscopy and real-time PCR. To test this technique, it was deployed to examine the deposition of
aerosolized fecal bacteria at various sampling sites with expected differences in bacterial population. The
selected sites, chosen to provide distinct bioaerosol compositions, consisted of a human fecal source near
the aeration basin at a municipal wastewater treatment facility, a cattle source barn-side at a dairy farm
(immediately next to a manure storage lagoon), and background at a remote camp in the northern
Adirondack Mountains. At each location, total ambient bacteria concentrations and population viability
were determined, and deposited bioaerosols screened for fecal indicator species and host-specific PCR
biomarkers. The goal of the study was thus to design analytical techniques able to discern expected
population differences with respect to these parameters, demonstrating the method’s utility in collecting
and analyzing deposited bioaerosols without the need for cultivation.
Methods
Bioaerosol samples for the study were taken at each of three predetermined locations. At each,
both the static water surface sampler (SWSS) and an open-face filter (OFF) collected two sets of sevenMichael Jahne, Environmental Engineering 2009; Dr. Shane Rogers, CEE Department
2008 REU in Environmental Sciences and Engineering at Clarkson University
hour samples. The SWSS (Figure 1) consists of a Petri dish seated within a large sharp-edged Plexiglas
deposition plate that minimizes air stream disruption, thus creating uniform laminar flow across its liquid
collection medium (Sahu et al., 2005). In doing so, this provided a
measure of dry deposition flux, with all bacteria collected assumed to
have been directly deposited vertically. To measure average overall
ambient concentration, the OFF used a vacuum pump assembly to draw
air through polycarbonate filters at a known flow rate. Environmental
parameters (wind speed and direction, temperature, and relative
humidity) were monitored throughout all sampling.
Analysis of samples from each collection method employed both
fluorescent microscopy using the LIVE/DEAD BacLight assay
(Molecular Probes, Inc., Eugene, OR) and real-time PCR. The
LIVE/DEAD kit uses two nucleic acid stains to distinguish between
“live” cells with intact membranes and “dead” membrane-compromised
cells, thus providing a measure of not only total cell count, but also an
indication of bacterial viability. For the SWSS, two slides were prepared
for each sample by filtering 50mL of collection buffer through a black
polycarbonate filter; cells collected on black polycarbonate filters in the
OFF were stained directly. Prepared slides were viewed at 1250X
magnification and 20 images per slide acquired for cell counting. The
remaining SWSS media was filtered onto a white polycarbonate filter
and, along with a filtered field blank and additional white polycarbonate
Figure 1: SWSS
filter from the OFF, retained for DNA extraction using the Ultra Clean
DNA Isolation Kit as per manufacturer’s instructions (Mo Bio
Laboratories, Inc., Solana Beach, CA). Eluted template DNA was then amplified by real-time PCR on a
Roche LightCycler 480 system (F. Hoffmann-La Roche, Ltd., Basel, Switzerland) to screen for target
sequences using primer/probe sets reported in the literature (Table 1) that included Domain-level probes
as well as those for fecal indicator species and host-specific PCR biomarkers. Salmon testes DNA was
used as an exogenous extraction control.
Table 1: Specific PCR Targets
Target
General Bacteria
Fecal indicators:
Enterococcus spp.
Fecal Bacteroidales
Host-specific PCR biomarkers:
Cattle/Ruminant
Human
GOI
16S rDNA
Reference
Nadkarni et al., 2002
ECST748
16S rDNA
Ludwig and Schleifer, 2000
Dick and Field, 2004
M2
CF128
HF183
Shanks et al., 2008
Bernhard and Field, 2000
Bernhard and Field, 2000
Preliminary Results
Results from the LIVE/DEAD assay (Table 2) indicate elevated bioaerosols at the wastewater
treatment plant and dairy farm relative to the Adirondacks camp as expected, with ambient populations at
the farm well above both other sites. Dry deposition onto the SWSS followed a similar trend, with
greatest flux seen at the farm site and flux near the wastewater facility above Adirondack background
level.
Michael Jahne, Environmental Engineering 2009; Dr. Shane Rogers, CEE Department
2008 REU in Environmental Sciences and Engineering at Clarkson University
Table 2: LIVE/DEAD Results
% Live
OFF Site Averages:
WWTP
Farm
Adk Mtns
SWSS Site Averages:
WWTP
Farm
Adk Mtns
Deposition Flux
(#/m2-hr)
Ambient Air Concentration
(#/m3)
45%
34%
71%
-
1.55E+05
4.05E+05
9.32E+04
30%
24%
69%
2.84E+07
1.04E+08
1.51E+07
-
These values correspond to reported literature measurements, including those found by Sahu et al.
(2005) using the SWSS near a wastewater treatment plant. In that study, atmospheric populations ranged
between 1.3 x 105 #/m3 and 1.4 x 105 #/m3 and flux between 7.7 x 106 #/m2-hr and 1.4 x 107 #/m2-hr.
Bacterial viability was greatest at the remote Adirondack site with a statistically significant increase to
69% “live” seen with the SWSS over the treatment plant and farm; viability using the OFF was
comparable (Table 2). Results of real-time PCR analyses relative to viable populations will also be
presented.
Although further sampling to improve authority of these findings is necessary, results tentatively
suggest an increase in both ambient bioaerosol concentration and deposition flux near fecal waste sites,
with particular elevation surrounding farming operations. Such a conclusion has adverse implications for
farm workers and their families, presenting the possibility of degraded public and environmental health
through both immediate air quality degradation and downwind deposition of aerosolized bacteria onto
water bodies and sensitive land-use areas. Additional work to continue into the fall of 2008 includes
supplementary sample sets from targeted sites, as well as side-by-side measurements using other
monitoring equipment. Broadcast manure spreading has also been identified as an additional source of
interest to be examined via edge-of-field sampling during manure application, and extended PCR analysis
using retained sample DNA will include screening for selected bacterial pathogens and antibioticresistance genes. The results of this and future studies will thus be useful to further examine the impact
human activities such as wastewater treatment and large-scale farming operations have on ambient
bioaerosol populations, their downwind deposition, and potential risks to receptor populations near these
facilities.
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Michael Jahne, Environmental Engineering 2009; Dr. Shane Rogers, CEE Department
2008 REU in Environmental Sciences and Engineering at Clarkson University
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Michael Jahne, Environmental Engineering 2009; Dr. Shane Rogers, CEE Department
2008 REU in Environmental Sciences and Engineering at Clarkson University