This document discusses a new approach to monitoring bioaerosols in Ireland using a real-time fluorescence instrument called the Waveband-Integrated Bioaerosol Sensor (WIBS). It describes how the WIBS can detect different types of primary biological aerosol particles (PBAPs) like pollen, fungi and bacteria based on their intrinsic fluorescence. Field tests of the WIBS in a rural area of Ireland showed that its measurements of fluorescent particles correlated well with traditional spore sampling, helping to identify the sources and behavior of various airborne biological particles.

1. A New Approach to Bioaerosol
Monitoring in Ireland
Summary
1. Particulate Matter (PM) in the Atmosphere
2. Primary Biological Atmospheric Particles (PBAP)
3. BioCheA: EPA 2007 CCRP Project 4.4.6.b
4. Where To and What Next? Future Applications of
the WIBS Technology
Professor John Sodeau
Department of Chemistry and Environmental Research Institute
University College Cork

2. ACKNOWLEDGEMENTS
Dr David Healy
David O’Connor (BIO-POSTER)
Dr Stig Hellebust
Ian O’Connor (CHEM-POSTER)
Dr Paul O’Driscoll
EPA for funding via BioCheA (2007 CCRP Project 4.4.6.b)
and a Doctoral Scholarship to David O’Connor
David Dodd

3. Airborne particles range in size, chemical (and biological)
composition and morphology. Some might be mainly
composed of sea-salt whereas others might contain toxic
transition metals such as cadmium in bioavailable forms.

4. PM contains biological components
Approximate chemical composition of
URBAN fine Particulate Matter (PM2.5)
The UCC group have
monitored PM10 and PM2.5
composition, EC/OC splits,
sulfates, SO2, ozone, NOX as
well as atmospheric Hg in
Cork harbour as part of a
variety of air quality
receptor-modelling
projects funded by EPA, EU
and SFI since 2006.
FUNGAL CELLS/SUB-POLLEN:
CONTRIBUTE 12-22% TO ORGANIC COMPOUNDS,OC
RESPONSIBLE FOR 4-11% OF TOTAL MASS

5. Primary Biological Aerosol Particles
PBAP
~0.5-3.0 (-30) mm
~0.02 – 0.5 mm
BACTERIA
VIRUSES
FUNGAL SPORES
POLLEN ~1.5-4.0 (-30) mm
~10-100 mm
PLANT ALGAE
DEBRIS
CRYPTOSPORIDIUM
OOCYSTS
~4-6 mm

6. On-line monitoring of PBAP
for the EPA
EPA
CCRP/STRIVE
2006-2010
REPORT
(2012)
Analysis of the development & occurrence
of
BIOlogical & CHEmical Aerosols (2008-2011)

7. Why monitor PBAPs in the Atmosphere?
Can act as ice
Pollen and fungal
nuclei and
spores can cause
initiate ice-crystal
allergies such as
formation in
hayfever
clouds
1. HEALTH 2. CLIMATE
Irish potato famine
Anthrax attacks in due to the fungus,
USA, 2001 Phytophthora
infestans
3. TERRORISM 4. WELFARE

8. Traditional PBAP Characterization:
Many Days of Intensive Labour
Impaction “Eyeball”
analysis
SporeWatch Optical
Microscopy
POLLEN

9. However PBAP fluoresce intrinsically
PBAP are known to fluoresce because they contain
intrinsic bio-fluorophores such as:
Trytophan, NAD(P)H and the Flavinoids
We utilize this property in our real-time detection method

10. The fluorescence spectra can be measured
Excitation at 370 nm
Grasses
1.40E-07
1.20E-07
Chlorophyll-a peak
1.00E-07
Fluorescence 8.00E-08
6.00E-08
4.00E-08
2.00E-08
0.00E+00
400 450 500 550 600 650 700
Wavelength (nm)

11. Collection of the fluorescence on-line
•Single particles analysed by a compact device
•Excitation at both 280 nm and 370 nm
•Fluorescence captured at (300-400 &
420-650 nm)
•Diode laser gives scatter signal to allow
determination of particle size (0.5-30 mm) and
asymmetry factor (“shape”), the AF value
•Time-resolution of secs-msecs REAL-TIME

12. Waveband-Integrated Bioaerosol Sensor
Aerosol in
Diode FL1
Laser (~300-
635nm 400nm)
Xe1
(280nm)
Forward
scatter
Side scatter
FL2 6°-25°
54°-126° Beam
(~420-650nm)
dump
Xe2
(370nm)
Copyright: DrW Stanley

13. WIBS 4 Real-Time Data Display:
Differing PBAP show differing
patterns of the 5 signals An alarm can be
depending on identity incorporated when
“high” levels of a
chosen signal are
exceeded
FL1 SHAPE
FL2
FL3
SIZE
SHAPE PARTICLE
TYPE

14. Individual PBAP have WIBS “signatures”
Optical Microscopy cannot
distinguish between these
important compost
bioaerosols
Normalised fluorescence laboratory measurements of FL1 and FL3 plotted
against optical particle size (median values) for each sample type coloured
according to its corresponding AF value. (The AF colour-bar indicates:
navy/blue as a particle that is more spherical in shape and red-brown
represents a more rod-like particle).

15. WIBS-4 Field Testing:
Real-time vs Traditional
Killarney National Park
(KNP)
August 2010
 Rural Setting: environment with
very low contributions from human
activities and anthropogenic
sources
 Four week campaign
 WIBS-4, SporeWatch, Weather
Station etc deployed

16. WIBS-4: Filtering the Data
(MILLIONS of particles observed in the campaign)
SET THRESHOLDS:
•Instrumental lower limit of fluorescence defined for
each FL1, FL2 and FL3
•Defined fluorescence thresholds
•Power performance of the lamps
OTHER FILTERS: IGOR Pro
(Wavemetrics)
• Size range, e.g. ignore > 20 mm
• By asymmetry, e.g. spherical or rod-like?
• Fluorescence signals, normalised to their FL ratios
• Campaign Site
Examples: Dust particles are large but non-fluorescent;
oil (PAH) droplets are fluorescent but small and spherical

17. Focus Period: 24 to 27-August-2010
• Hourly median values vs time of day PLUS Relative Humidity Data
Some Conclusions
• Sum of Ascospores, FL3
• Following diurnal trend of fluorescent particles in channels FL2 &Badiospores
& Ganoderma fungal spore
numbers track well the diurnal
trends observed in FL2 and FL3
FL2-SIZE
• Fungal spores appear at night-
time when 80-100 %RH
• FL2 data indicate two PBAP
FL3-SIZE
groups (~1.2 mm and ~ 3 mm:
median values); former not
pronounced in FL3 and latter not
pronounced in FL1)
SporeWatch
2
2.5x10
Trap Data
• ~0.8-1.5 mm group:
2.0
Ganoderma
Concentration (m-3)
1.5
Basidiospores
1.0
Ascospores
Actinomycetes / Streptomycetes
0.5
NFL3 from soil?
0.0
0 5 10 15 20
Time of Day (hr)
4

18. BioChea Sampling Campaigns
Uni. Of
Killarney Hertfordshire
National
Park
Port of Cork Karlsruhe
Munich

19. Where To and What Next?
Future applications of the WIBS
technology in light of BioCheA
discoveries

20. WASTE MANAGEMENT
Planning and permitting bodies such
as councils and the Environment
Agency (EA) now require risk
assessments and regularly monitor
bioaerosol emissions on sites that fall
under the "potential to cause local
health concern to the workers and
surrounding public" category of Current preferred method
facilities/sites. How easy is this employs direct impaction
currently? (Anderson Sampler) where
Petri dishes of appropriate
The British Standards Institute (BSI) media are loaded into the
recognises the increasing levels of sampler. The dishes are then
concern and interest in bioaerosols incubated in a laboratory and
and has released a technical the bacteria/fungal spore
specification (CEN/TS 16115-1:2011) colonies (CFU) counted later
on the measurement of moulds in by optical microscopy
ambient air to identify, quantify and Not real time data:
characterise bioaerosol pollution in “snapshot” sampling.
ambient air resulting from emissions Impactor can become
from different sources. overloaded quickly

21. OCCUPATIONAL LUNG DISEASES
Farmers’ lung and Aspergillosis are
caused by the inhalation of thermophilic
Actinomycetes (0.5-1.5 mm) or Aspergillus
(2-3.5 mm) species in decomposing
compost, hay, or sugar cane. Exposure to
large quantities of contaminated hay is
the most common source. This most
commonly occurs during the winter
months due to the cold, damp climate.
Incidence is highly variable but it is
estimated to affect 0.4%-7% of the
farming population.

22. WIBS can distinguish between PBAP in HAY using SIZE
2.5 mm
Previous studies show that
2.0 mm the size range for Aspergillus
3.5 mm
AND Penicillium is 2-3.5 mm.
Two PBAP clusters of
similar size (2- 3 mm) and
8
2.0
similar FL2:FL3 ratio but 6
1.5
differing FL1:FL3 ratio.
FL2/FL3
1.0
FL1/FL3
Cluster ~0.5-1.5 mm with 4
0.5
different fluorescence 2 0.0
character from both the
other two clusters:
Actinomycetes? 2 4
Size (microns)
6 8

23. WIBS can distinguish between PBAP in HAY using SHAPE
AF values closer to 0 indicate
SPHERICAL shape. Closer to
100 indicates ROD-LIKE shape.
Aspergillus and Penicillium are
close to spherical/ovoid.
Two PBAP clusters of 8
5
similar size (~2 mm) and 6
4
Size (microns)
spherical shape particles 3
FL1/FL2
but differing FL1:FL2 and 4 2
FL2:FL3 ratios 2
1
0
Cluster includes smaller
sizes (~1 mm) with FL ratios
20 40 60 80
AF (shape factor)
Our optical
similar to one of the larger microscope
picture of
clusters: Actinomycetes?
hay “dust”

24. IRRITANTS AND KILLERS
The annual "Pollen Count" for Ireland
shown on the Irish Health PollenAlert app is
compiled entirely from monitoring
measurements made in the UK. They are
sold to us as a computer model prediction
by the University of. Worcester. An outbreak of Legionaires Disease
Although Ireland was a pioneer in this field broke out in Scotland in early June
many years ago, the labour intense nature 2012. It was ascribed to the
of the work coupled with the high level of aerosolized release of Legionella
expertise required using the traditional pneumonia, a bacterium that
impaction/microscopy approach has meant possesses a distinctive rod-like
that we no longer provide a service for shape. (0.5-0.7 x 2 mm long)
ourselves. Poolbeg power station, for example,
Can the WIBS provide an on-line approach has such water coolers. If Dublin
to supply real-time measurements of the were ever to experience a similar
pollen count? outbreak, could WIBS help to provide
a rapid, on-line, on-site analysis?
And what about cryptosporidium oocysts (4-6 mm)
aerosolized release from contaminated water and
"biosolids"?

25. Pollen event in the Yew Forest at KNP:
February 2010
WIBS 4
Pollen Event Date Start time Finish time
Seconds
resolution 28/02/2010 11:44.49 16:26.55
28/02/2010 SporeWatch Data SporeWatch
Yew counts hazel counts alder counts
700
Pollen Counts (grains per m3)
600
500
16.30
400
11.45
2 hour Pollen event.....is mainly
300
resolution YEW pollen
200
100
0

26. WIBS can measure sizes up to 30 mm:
Yew pollen Yew pollen size
distribution
100
Particle Count
50
0
18 19 20 21 22 23 24 25 26 27 28 29 30 31
Size AF TOF FL1_280 FL2_280 FL2_370
Mean 26.70 18.75 8.75 973.17 2044.73 1954.31
Median 26.85 17.57 8.75 892.00 2077.00 1967.00 AF
200
Mode 30.55 19.86 8.94 2116.00 2077.00 1967.00
Standard 150
frequency
Deviation 2.60 7.95 0.67 398.42 141.47 109.98
100
Sample
Variance 6.78 63.18 0.45 158741.33 20012.89 12096.67 50
Kurtosis -0.32 0.52 2.01 0.35 50.59 169.67 0
5 15 25 35 45 55
Skewness -0.36 0.65 -0.34 0.73 -6.36 -12.27 AF values
Range 11.94 48.27 5.56 2094.00 1706.00 1713.00
Minimum 18.61 1.01 5.46 22.00 371.00 254.00
Maximum 30.55 49.27 11.02 2116.00 2077.00 1967.00
Count 582 582 582 582 582 582

27. INDOOR AIR
Problems of indoor air quality are
recognized as important risk factors for
human health. In hospitals, day-care
centres, retirement homes and schools,
microbial air pollution affects population
groups that are particularly vulnerable.
In a given space, concentrations of fungal
spores (Aspergillus/Penicillium) in indoor
environments are highly variable and depend
upon climate, season and the sampling
methods employed, which make studies to
date difficult to compare with any validity.
Pseudomonas aeroginosa
Pseudomonas baby
infection deaths in
Belfast's Royal
Jubilee Hospital,
January 2012

28. WIBS studies from BioCheA on PSEUDOMONAS
Real-time detection of bacteria pulse
Ice nucleation experiments
performed at the AIDA
chamber in Germany
• Bacterial species isolated from
cloud water
(Pseudomonas Do~ 0.7 mm in
“diameter”)
• Sampled from Puy de Dôme
station (Clermont-Ferrand,
France)

29. BIOCHEA RESULTS SHOW:
The WIBS technique offers a comprehensive, one-stage, real-
time, remotely operated monitoring methodology for
bioaerosols of environmental concern both outdoors (and
indoors):
1. It discriminates between chemical and biological aerosols.
2. It discriminates between bioaerosols by size and “shape”
and their fluorescence characteristics.
3. It can distinguish between bioaerosols that cannot be
distinguished by optical microscopy.
4. It has a high throughput: up to 125 particles per sec
5. It employs cheap, robust xenon flash-lamp sources used in
cameras and a diode laser rather than expensive laser
excitation sources used in earlier and alternative
approaches.

30. THANK YOU

31. Question and Answer Slides

32. Particulate Matter (PM)
PM is a complex mixture of extremely small particles and liquid droplets.
Chemically it comprises a number of components including acids (such
as nitrates and sulfates), organic compounds (OC), elemental carbon
(EC), transition metals, soil, fugitive dust and sea-spray.
Fine fraction Coarse fraction
(PM2.5) Elemental and
(PM2.5-PM10) PM size is directly
Organic Carbon
Sulphate linked to their
Nitrate
potential for
Ammonium
Chloride
causing health
Insoluble minerals problems. The
Na, K, Mg, Ca smaller the more
Approximate chemical composition of lethal.
Particulate Matter

33. Overview of WIBS operating principles
Single particle measurements
Size:
~0.5 µm to 12 μm
~3 µm to 31 μm
Index of shape:
Uses scatter intensity values received
by each quadrant of a quadrant PMT
detector to calculate an AF value
Wide Issue Fluorescence
FL1:
Bioaerosol Sensor 280 nm excitation; emission ~310 – 400 nm
(WIBS)
FL2:
Combines particle UV fluorescence, 280 nm excitation ; emission ~420 – 650 nm
particle sizing & ‘shape’
assessment in one sensor FL3:
370 nm excitation; emission ~420 – 650 nm

34. Overview of the Killarney National Park
Campaign
SPOREWATCH DATA
FL1 λex = 280nm
λem = ~310 – 400 nm
FL2 λex = 280nm
λem = ~420 – 650 nm
FL3 λex = 370nm
λem = ~420 – 650 nm
0.5-12 mm
range of
Focus Period sizes

35. February 2010 campaign in KNP:
Particles > 20μm
NF (cm-3)
1 2
“Pollen” events