real time detection of airborne fungal spores and ... · iv commercially available instrument...
TRANSCRIPT
QUEENSLAND UNIVERSITY OF TECHNOLOGY
SCHOOL OF PHYSICAL AND CHEMICAL SCIENCES
REAL TIME DETECTION OF AIRBORNE
FUNGAL SPORES AND INVESTIGATIONS INTO
THEIR DYNAMICS IN INDOOR AIR
A Thesis submitted by Hussein Fadhl Remathie Kanaani to the School of
Physical Sciences, Queensland University of Technology, in partial
fulfilment of the requirements of the degree of Doctor of Philosophy.
August 2009
ii
Keywords
Fungal spores; real-time monitoring; counting efficiency; spore release; air flow rate;
culturing time; fluorescent percentage; spore size; fragments; air velocity; deposition rate;
air exchange rates (AER); fungal particles; ventilation rate.
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Abstract
Concern regarding the health effects of indoor air quality has grown in recent years, due
to the increased prevalence of many diseases, as well as the fact that many people now
spend most of their time indoors. While numerous studies have reported on the dynamics
of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly
understood and very few studies have focused on fungal spore dynamics in indoor
environments. Consequently, this work investigated the dynamics of fungal spores in
indoor air, including fungal spore release and deposition, as well as investigating the
mechanisms involved in the fungal spore fragmentation process. In relation to the
investigation of fungal spore dynamics, it was found that the deposition rates of the
bioaerosols (fungal propagules) were in the same range as the deposition rates of non-
biological particles and that they were a function of their aerodynamic diameters. It was
also found that fungal particle deposition rates increased with increasing ventilation rates.
These results (which are reported for the first time) are important for developing an
understanding of the dynamics of fungal spores in the air. In relation to the process of
fungal spore fragmentation, important information was generated concerning the airborne
dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation.
The results obtained from these investigations into the dynamics of fungal propagules in
indoor air significantly advance knowledge about the fate of fungal propagules in indoor
air, as well as their deposition in the respiratory tract.
The need to develop an advanced, real-time method for monitoring bioaerosols has
become increasingly important in recent years, particularly as a result of the increased
threat from biological weapons and bioterrorism. However, to date, the Ultraviolet
Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only
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commercially available instrument capable of monitoring and measuring viable airborne
micro-organisms in real-time. Therefore (for the first time), this work also investigated
the ability of the UVAPS to measure and characterise fungal spores in indoor air. The
UVAPS was found to be sufficiently sensitive for detecting and measuring fungal
propagules. Based on fungal spore size distributions, together with fluorescent
percentages and intensities, it was also found to be capable of discriminating between two
fungal spore species, under controlled laboratory conditions. In the field, however, it
would not be possible to use the UVAPS to differentiate between different fungal spore
species because the different micro-organisms present in the air may not only vary in age,
but may have also been subjected to different environmental conditions. In addition,
while the real-time UVAPS was found to be a good tool for the investigation of fungal
particles under controlled conditions, it was not found to be selective for bioaerosols only
(as per design specifications). In conclusion, the UVAPS is not recommended for use in
the direct measurement of airborne viable bioaerosols in the field, including fungal
particles, and further investigations into the nature of the micro-organisms, the UVAPS
itself and/or its use in conjunction with other conventional biosamplers, are necessary in
order to obtain more realistic results.
Overall, the results obtained from this work on airborne fungal particle dynamics will
contribute towards improving the detection capabilities of the UVAPS, so that it is
capable of selectively monitoring and measuring bioaerosols, for which it was originally
designed. This work will assist in finding and/or improving other technologies capable of
the real-time monitoring of bioaerosols. The knowledge obtained from this work will also
be of benefit in various other bioaerosol applications, such as understanding the transport
of bioaerosols indoors.
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List of publications
Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia Morawska (2007).
Performance assessment of UVAPS: Influence of fungal spore age and air exposure.
Journal of Aerosol Science 38: 83-96.
Hussein Kanaani, Megan Hargreaves, Jim Smith, Zoran Ristovski, Victoria Agranovski
and Lidia Morawska (2008). Performance of UVAPS with respect to detection of
airborne fungi. Journal of Aerosol Science 39: 175-189.
Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia Morawska (2008).
Deposition rates of fungal spores in indoor environments, factors effecting them and
comparison with non-biological aerosols. Atmospheric Environment 42: 7141-7154.
Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia Morawska. Fungal spore
fragmentation as a function of airflow rates and fungal generation methods. Has been
accepted for publication in ‘Atmospheric Environment’ in April 2009.
vi
Table of contents
Keywords…………………………………………………………………………………………………………………….ii
Abstract……………………………………………………………………………………………………………………..iii
List of publication………………………………………………………………………………………………………..v
Table of contents………………………………………………………………………………………………………..vi
List of Tables................................................................................................................... xv
List of Figures................................................................................................................. xvi
CHAPTER 1 : Introduction ............................................................................................. 1
1.1 A description of scientific problem investigated ..................................................1
1.2 Overall aims of the project....................................................................................3
1.3 Specific objectives of the study.............................................................................4
1.4 Account of scientific progress linking the scientific papers ..................................5
References........................................................................................................................9
CHAPTER 2 : LITERATURE REVIEW............................................................................... 12
2.1 Pollutants, aerosols and bioaerosols in indoor environments ...........................12
2.1.1 Aerosols ...........................................................................................................13
2.1.2 Bioaerosols ......................................................................................................14
2.1.2.1 Concentration of fungal spores in indoor and outdoor environments ...14
2.1.3 Fungi ................................................................................................................16
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2.1.3.1 Definition and taxonomy..........................................................................16
2.1.3.2 Structure...................................................................................................18
2.1.3.3 Fungal spores: size, shape and density ....................................................18
2.1.3.4 Growth and reproduction ........................................................................20
2.1.3.5 Fungi and moisture ..................................................................................22
2.1.3.6 Fungal fragmentation...............................................................................23
2.1.3.7 Fungal secondary products ......................................................................24
2.1.3.8 Microbial growth prevention ...................................................................27
2.1.3.9 Benefits and impacts of fungi...................................................................28
2.1.3.9.1 Benefits................................................................................................28
2.1.3.9.2 Impacts ................................................................................................29
Impact on Human Health ................................................................................29
Impact on Animals, Plants and Building Materials..........................................31
2.1.4 Summary..........................................................................................................32
2.2 Fungal spore dynamics in indoor air ...................................................................34
2.2.1 General Introduction .......................................................................................34
2.2.1.1 Ventilation and its effects on fungal spore deposition rates...................36
2.2.1.1.1 Ventilation ...........................................................................................36
Fungal Spore Deposition Rates........................................................................38
2.2.2 Fungal Release .................................................................................................40
2.2.3 Fungal spore transport ....................................................................................42
2.2.4 Interaction of spores with non‐biological aerosols.........................................43
2.2.5 Deposition........................................................................................................44
2.2.6 Summary..........................................................................................................46
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2.3 Aerosol and bioaerosol sampling techniques and samplers...............................48
2.3.1 Introduction.....................................................................................................48
2.3.2 Sampling methods ...........................................................................................49
2.3.3 Bioerosal air samples: air samplers and techniques .......................................49
2.3.3.1 Conventional methods of bioaerosol air sampling ..................................50
2.3.3.1.1 Inertial impaction ................................................................................51
2.3.3.1.2 Liquid impingement.............................................................................52
2.3.3.1.3 Filtration ..............................................................................................53
2.3.3.1.4 Electrostatic precipitator.....................................................................54
2.3.3.1.5 Gravitational sedimentation ...............................................................55
2.3.3.2 Real‐time methods in bioaerosol air sampling ........................................56
2.3.3.2.1 Fluorescence and biochemical fluorophores ......................................57
2.3.3.2.2 Organism viability and the natural auto‐fluorescence........................58
2.3.3.2.3 Ultraviolet Aerodynamic Particle Sizer (UVAPS) .................................60
2.3.4 Summary..........................................................................................................63
2.4 Knowledge Gaps..................................................................................................64
References......................................................................................................................67
CHAPTER 3 : Performance of UVAPS with Respect to detection of airborne fungi....... 91
Statement of Joint Authorship .......................................................................................92
Abstract ..........................................................................................................................93
3.1 Introduction.........................................................................................................94
3.2 Materials and methods .......................................................................................96
ix
3.2.1 The UVAPS calibration.....................................................................................96
3.2.2 Sample preparation .........................................................................................97
3.2.2.1 Fungal species used for aerosolization by Collison nebulizer..................97
3.2.2.2 Fungal species used for aerosolization by dry (direct) method...............97
3.2.3 Microscopic analysis ........................................................................................98
3.2.4 Experimental set up and procedures ..............................................................98
3.2.4.1 Generation of fungal aerosols with a Collison nebulizer .........................98
3.2.4.2 Dry (direct) spore generation method.....................................................99
3.2.4.3 Operational procedures .........................................................................100
3.3 Results and Discussion ......................................................................................102
3.3.1 The UVAPS calibration...................................................................................102
3.3.2 Application of Collison nebulizer for generating fungal aerosols .................104
3.3.3 Direct generation method .............................................................................107
3.3.3.1 Fungal particles morphology..................................................................107
3.3.3.2 Fungal spore aerosolization ...................................................................108
3.3.3.3 UVAPS and AGI 30 correlation ...............................................................113
3.3.3.4 The potential of the UVAPS in differentiating between fungal species 117
3.3.3.5 The relationships between total and fluorescent particle concentrations
................................................................................................................119
3.4 Conclusions........................................................................................................122
Acknowledgements......................................................................................................123
References....................................................................................................................124
x
CHAPTER 4 : Performance assessment of UVAPS: Influence of fungal spore age and air
exposure ...................................................................................................................128
Statement of Joint Authorship .....................................................................................129
Abstract ........................................................................................................................130
4.1 Introduction.......................................................................................................131
4.2 Materials and Methods.....................................................................................134
4.2.1 Aerosol preparation.......................................................................................134
4.2.2 Apparatus description ...................................................................................135
4.2.3 Experimental Methodology...........................................................................136
4.2.3.1 UVAPS calibration...................................................................................137
4.2.3.2 The fungal spore size and fluorescent percentage as a function of fungal
age ..........................................................................................................138
4.2.3.3 Fungal spore fluorescent percentage as a function of the frequency of
exposure.................................................................................................139
4.2.3.4 Fungal particle identification .................................................................140
4.3 Results ...............................................................................................................141
4.3.1 Fungal particle identification.........................................................................141
4.3.2 Fungal spore size ...........................................................................................141
4.3.3 Fungal spore fluorescence as a function of culturing time ...........................144
4.3.4 Fluorescent Intensity .....................................................................................145
4.3.5 Fungal spore fluorescent percentage as a function of frequency of the
exposure ........................................................................................................150
4.4 Discussion..........................................................................................................151
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4.4.1 Fungal spore size as a function of age...........................................................152
4.4.2 The effect of age on fungal spore fluorescent percentage ...........................152
4.4.3 The effect of age on the fungal spore fluorescent intensity ........................154
4.4.4 The effect of air current on spore fluorescent percentage...........................155
4.5 Conclusions........................................................................................................156
Acknowledgements......................................................................................................157
References....................................................................................................................158
CHAPTER 5 : Fungal spore fragmentation as a function of airflow rates and fungal
generation methods ................................................................................................. 163
Statement of Joint Authorship .....................................................................................164
Abstract ........................................................................................................................165
5.1 Introduction.......................................................................................................166
5.2 Materials and methods .....................................................................................168
5.2.1 Fungal preparation ........................................................................................168
5.2.2 Instrumentation and applied protocol ..........................................................169
5.2.3 Fungal aerosol generation methods..............................................................170
5.2.3.1 Generation of fungal aerosols by the direct method ............................171
5.2.3.2 Generation of fungal aerosols using the fan method ............................171
5.2.3.3 Generation of fungal particles using the FSSST method........................171
5.2.4 Data analysis ..................................................................................................174
5.3 Results and Discussion ......................................................................................174
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5.3.1 Related fungal fragment particles as a function of air velocity and generation
methods.........................................................................................................174
5.3.2 The characterisation and mechanism of fungal spore fragmentation..........182
5.3.3 The expected impact of spore fragmentation on health ..............................190
5.3.4 The correlation between spores and fragment release................................193
5.3.5 Fungal particle fluorescent percentage.........................................................193
5.3.6 Comparison of size distribution of fungal fragmentation particles as measures
by UVAPS and SMPS ......................................................................................195
5.4 Conclusions........................................................................................................198
Acknowledgements......................................................................................................199
References....................................................................................................................200
CHAPTER 6 : Deposition rates of fungal spores in indoor environments, factors
effecting them and comparison with non‐biological aerosols. ...................................205
Statement of Joint Authorship .....................................................................................206
Abstract ........................................................................................................................207
6.1 Introduction.......................................................................................................208
6.2 Material and Methods ......................................................................................209
6.2.1 Experimental chamber and mechanical ventilation system .........................209
6.2.2 Air speed, air flow pattern, temperature and humidity monitoring.............211
6.2.3 CO2 measurements and air exchange rate (AER) estimation........................212
6.2.4 The UVAPS instrument .................................................................................213
6.2.5 Sample preparation .......................................................................................214
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6.2.6 Aerosol generation and measurement .........................................................215
6.2.7 Fungal particle identification.........................................................................217
6.2.8 Estimation of particle deposition rates .........................................................218
6.2.8.1 Ventilation system off ............................................................................218
6.2.8.2 Ventilation system on ............................................................................219
6.3 Results ...............................................................................................................220
6.3.1 Air exchange rates, airspeed and airflow pattern.........................................220
6.3.2 Deposition rate with the ventilation system off ...........................................223
6.3.3 Deposition rates with the ventilation system on ..........................................226
6.3.4 Aerosol fluorescent percent..........................................................................230
6.4 Discussion..........................................................................................................231
6.4.1 Air exchange rates, air speed and airflow patterns ......................................231
6.4.2 Fungal spore concentration levels.................................................................233
6.4.3 Deposition rates and comparison with other studies when the ventilation
system was off ...............................................................................................233
6.4.4 Deposition rates and comparison with other studies when the ventilation
system was on ...............................................................................................236
6.4.5 Aerosol fluorescent percent..........................................................................240
6.5 Conclusion .........................................................................................................241
Acknowledgements......................................................................................................242
References....................................................................................................................243
CHAPTER 7 : General Discussion................................................................................ 249
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7.1 Introduction.......................................................................................................249
7.2 Principal significance of the findings.................................................................250
7.3 Conclusions........................................................................................................255
7.4 Future work .......................................................................................................256
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List of Tables
Table 3‐1: Tested fungal characteristics.................................................................................................... 108
Table 3‐2: Aerosol results as measured by UVAPS and impingers............................................................ 117
Table 4‐1: Average percentage of different sizes of fungal spores at different ages: (a) Aspergillus; (b) Penicillium......................................................................................................................... 143
Table 4‐2: Fluorescent percentage of fungal spores (Aspergillus and Penicillium) after seven days culturing time ............................................................................................................................................ 146
Table 5‐1: Percentage of fungal fragmentation as a function of air velocity and generation methods..................................................................................................................................................... 180
Table 5‐2: Typical samples of fungal spores under investigation; before and after fragmentation......... 187
Table 5‐3: Fluorescent percentage of fungal species with and with out fragmentation (fan method). .................................................................................................................................................... 195
Table 6‐1: Standard deviations for particle deposition rate coefficients (h‐1) (which are presented in Figure 6.4) as measured at different air exchange rates....................................................................... 228
Table 6‐2: Particle deposition rate coefficients (h‐1), for each of the four categories .............................. 229
xvi
List of Figures
Figure 2.1: Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St, Paul, MN).........99
Figure 2.2: Aerosol flow through the UVAPS. ................................................................................99
Figure 3.1: Experiment set‐up (Collison nebulizer method). a, Flow Meter; b, Dryer; (c), Pressure equalizing holes. ......................................................................................................99
Figure 3.2: Experimental set‐up (direct method). (c), Pressure equalizing holes. .......................100
Figure 3.3: UVAPS spectra for blank and standards: (a) Blank (direct method). (b) PSL (0.993 µm) generated by Collison nebulizer.................................................................................103
Figure 3.3: UVAPS spectra for Blue fluorescent standards: (c) blue Fluorescent (0.91 µm) generated by Collison nebulizer ...................................................................................................103
Figure 3.4: UVAPS spectra (APS part) for aerosols generated by Collison nebulizer: (a) (SS‐2‐PXG 0.1) standard; (b) A mixture of (SS‐2‐PXG 0.1), (SS‐5‐PXG 0.1), (SS‐7‐PXG 0.1), (SS‐10‐PXG 0.1) and (SS‐15‐PXG 0.1) standards. ..........................................................................106
Figure 3.4: UVAPS spectra (APS part) for aerosols generated by Collison nebulizer: (c) Penicillium species (ACM4616). ....................................................................................................103
Figure 3.5: UVAPS spectra for fungal spores (direct method): (a) Penicillium sp.; (b) A. niger. .............................................................................................................................................110
Figure 3.5: UVAPS spectra for fungal spores (direct method): (c) Penicillium sp. (low concentration) ..............................................................................................................................116
Figure 3.6: UVAPS spectra (APS part) for fungal aerosol: (a) Penicillium sp.; (b) A. niger. ..........112
Figure 3.6: UVAPS spectra (APS data) for fungal aerosols: (c) Fragmentation of Penicillium sp. at air flow rate of 25 L/min ...................................................................................116
Figure 3.7: Fungal spore measurements of UVAPS and AGI‐30 impingers during the experiment investigating their correlation: (a) Penicillium sp.; (b) A. niger. ...............................116
Figure 3.8: The range of total and fluorescent particle concentrations from fungi as measured by UVAPS during the experiment investigating the correlation with AGI‐30 impingers. .....................................................................................................................................117
xvii
Figure 3.9: The range of total concentrations and fluorescent percentage for high fluorescent percentage cultures as measured by UVAPS, during the experiment investigating the correlation with AGI‐30 impingers....................................................................119
Figure 3.10: Fluorescent particles of Penicillium sp. as a function of total particles by UVAPS, similar trend was obtained for A. niger (data not shown) ..............................................121
Figure 4.1: Experimental set‐up for the UVAPS calibration, a, Flow Meter; b, Dryer; (c), Pressure equalizing holes..............................................................................................................137
Figure 4.2: Experimental set‐up (direct method generation), (c) Pressure equalizing holes..............................................................................................................................................139
Figure 4.3: Average spore size distribution spectra as a function of culturing time: (a) Aspergillus; (b) Penicillium; (c) Comparison of Aspergillus and Penicillium. ................................142
Figure 4.4: Fluorescent percentage as a function of culturing time of Aspergillus and Penicillium. ....................................................................................................................................144
Figure 4.5: Spore concentration percentage (all diameters in single channel) as distributed between UVAPS channels: (a) Aspergillus and Penicillium species after seven days culturing time; (b) Aspergillus after seven days and Penicillium after 14 days culturing time................................................................................................................................148
Figure 4.6: Average spore concentration percentage (for specific diameter in a single channel) of seven day old spores as distributed between UVAPS channels (channel numbers reflect degree of fluorescence): (a) Aspergillus (b) Penicillium.....................................150
Figure 4.7: Fungal spore fluorescent percentage as a function to number of times exposed to air flow rate of 10 L/min. ...........................................................................................151
Figure 5.1 Experimental set‐up for fungal aerosolisation: (a) Direct method; (c) Fan method; (b) Fungal spore source strength tester (FSSST) method. Remaining schematic drawing (biological safety cabinet, SMPS, UVAPS and HEPA filters) as in part (a).......................173
Figure 5.2 Typical UVAPS spectra for non‐fragmented (Top) and fragmented fungal species (Below): (a) Penicillium species (ACM 4616)....................................................................176
Figure 5.2: Typical UVAPS spectra for non‐fragmented (Top) and fragmented fungal species (Below): (b) Aspergillus niger (ATCC 9142) ......................................................................179
Figure 5.2: Typical UVAPS spectra for non‐fragmented (Top) and fragmented fungal species (Below): (c) Cladosporium cladosporoides (FRR 5106) ....................................................180
xviii
Figure 5.3 The frequency of fragmentation of 60 successive samples (20 seconds each) for different aerosolisation methods for Penicillium species. (The ‘No Frag.’ data are presented for comparison purposes and are derived from using the fan method at 5.3 m/s). ..............................................................................................................................................183
Figure 5.4: Average particle concentration (for all 10 high fluorescent percentage experiments) of fragmented and non‐fragmented fungal species for each of the three generation methods: ....................................................................................................................186
Figure 5.5: The particle concentration percentage of fungal particles with (right side) and with out (left side) fragmentation as detected by UVAPS: (a) Penicillium; (b) Aspergillus; (c) Cladosporium. ..........................................................................................................................192
Figure 5.6: Fluorescent percentages of Aspergillus (high fluorescent) particles as a function of particle diameters. .....................................................................................................195
Figure 5.7: Typical samples of Penicillium and Aspergillus detected and measured by SMPS and UVAPS, simultaneously. ...............................................................................................198
Figure 6.1: Experimental set‐up....................................................................................................211
Figure 6.2 Air speed and smoke flow pattern at V1: (a) three dimensional view of air speed flow at level 1.7m; (b) Top view of the smoke pattern flow..............................................222
Figure 6.3 Typical particle concentration as a function of elapsed time for aerosols and bioaerosols under investigation, at V0 air exchange rate conditions. ..........................................225
Figure 6.4 Particle deposition rate coefficient, as a function of aerodynamic diameter, for aerosols and bioaerosols, at different air exchange rates: (a) V0 (airspeed = 0.01 m/s); (b) V1 (airspeed = 0.15 m/s); (c) V2 (airspeed = 0.30 m/s). ................................................................226
Figure 6.5 Typical particle concentration profiles as a function of time for the aerosols under investigation at V1 air exchange rate. ................................................................................227
Figure 6.6 Typical particle fluorescent percentage as a function of time: (a) air exchange rate at V0 (AER = 0.009 ± 0.003 h
‐1); (b) air exchange rate at V1 (AER= 1.75 ± 0.32 h‐1)...............231
Figure 6.7 A comparisons of particle deposition rates measured in houses and experimental chambers reported in literature and those found in this study: (a) no ventilation, where V0 represents this study; (b) ventilation or fan on, where V1 represents this study. ...................................................................................................................239
xix
The Statement of Original Authorship
The work contained in this thesis has not been previously submitted for a degree or
diploma in any other higher education institution. To the best of my knowledge and
belief, the thesis contains no material previously published or written by any other person,
except where due reference is made.
Signed:
Date: 28 August 2009
xx
Acknowledgements
In the Name of Allah, the most Gracious, the most Merciful.
I wish to express my sincere gratitude, respect, honour and appreciation to my supervisor
Professor Lidia Morawska for her kindness, help, guidance and enthusiastic support, as
well as for giving me a chance to join her laboratory. Lidia has not only been my
supervisor but also my friend and I have learnt many things from Lidia for my
professional career.
I would also like to take this opportunity to thank Dr Megan Hargreaves, my co-
supervisor, for her great help, expertise and feedback, as well as for her guidance in
microbiological science. I am very grateful to Dr Zoran Ristovski who was not only co-
supervisor but also a friend, and he made an important contribution to this work,
especially in relation to the methods and techniques used in this study. I would also like
to take the opportunity to thank A/Prof Godwin Ayoko for being a co-supervisor and a
friend, as well as for the great effort he made in helping me to get this opportunity.
Special thanks go to the Research Triangle Institute for their grant support, and sincer
thanks go to Dr David Ensor, Aerosol Science and Nanotechnology, whose vision made
this study possible. The financial support Queensland University of Technology for one
year is highly appreciated.
Sincere thanks are extended to my colleagues and friends from ILAQH for their guidance,
wisdom and humour, especially Dr Nic Meyer, Dr Congrong He and Dr Graham Johnson
for their help in relation to instrument operation at the beginning of my study.
xxi
Special thanks go to Afkar, Mohammed, Yudi, Diane and Sade for being good friends
during my study, not only for their humour and their jokes, but also for their constructive
criticism. I have had a great time with them.
Special thanks also go to Ms Sue Gill, her staff and other associated staff in the School of
Life Science, for purchase and preparation of the fungal species, as well as for their
technical help operating the microbiological equipment and other tools used in this study.
Specifically, I would also like to thank Ms Sue Gill for her help, advice, kindness and
friendship, it is highly appreciated. Special thanks go to science workshop staff; Bob
Organ and Jim Drysdale for their effort in building what you want. The assistance
provided by the senior research support specialist, Mr Ray Duplock, in relation to the
statistical analysis performed in paper three and four of the study and that of the
coordinator research training Ms Kerry Kruger in relation to formatting the final shape of
the thesis also gratefully acknowledged. I would also like to thank Ms Rachael Appleby
for her help, administrative support and language corrections to both my papers, as well
as this thesis.
Special and sincere thanks go to my wife Sajida Albahadily for her faith, patience,
sacrifice and great support over the years. Special and sincere thanks go to my sons Ali,
Mohamed and Hassan and, as well as my dauhter Hawraa for their great understanding,
sacrifices, support and patience. Many thanks also go to my brothers (specially
Mohammed), sisters, relatives and friends, both here and overseas, and sincere thanks are
extended to everyone else who helped me in any way, whom I may have forgotten or
could not mention their name here.
1
CHAPTER 1 : Introduction
1.1 A description of scientific problem investigated
In indoor environments, where people spend most of their time (Ott, 1982; Lebowitz,
1983; Byrne, 1998; Brasche and Bischof, 2005), the presence of microbial contamination
has been confirmed by many studies around the world (Pei-Chih et al., 2000; Hargreaves
et al., 2003c; Portnoy et al., 2004; Jo and Seo, 2005).
Biological aerosols are released into the atmosphere from a variety of plant and animal
sources, including pollen, fungi and fungal spores, bacteria, viruses, domestic dust mites
and their excreta, algae, protozoa, toxins, fragments, volatile organic compounds, and the
particulate waste products of many microorganisms, plants and animals (Dudney and
Copenhaver, 1984; Maroni et al., 1995; Macher, 1999; Hobbs, 2000; Grinshpun and
Clark, 2005; Zhang, 2005).
Scientific and medical evidence has shown that exposure to biological aerosols can have
significant health implications, similar to those caused by other airborne pollutants (Dales
et al., 1991; Koskinen et al., 1997; Verhoeff and Burge, 1997a). For example, diseases
such as diphtheria, tuberculosis or legionellosis are often caused by airborne infectious
microorganisms (Macher, 1999; Maus et al., 2001).
Fungi and molds are considered to be potential health hazards (Fischer and Dott, 2003)
and the presence of fungal spores in the atmosphere is largely responsible for causing
respiratory distress in humans, as well as many allergic diseases such as asthma, rhinitis,
allergic bronchopulmonary mycoses and hypersensitivity pneumonitis (Vijay et al.,
1999b; Nester et al., 2004; Kim et al., 2007).
2
While indoor particle dynamics, including deposition and transport, are some of the most
important factors that determine the effect of particle exposure on human health, little is
known about the dynamics of biological aerosols in indoor environments, including
fungal spores. While a large number of studies have been conducted to determine the
deposition rates of non-biological aerosols in indoor environments (see Chapter 6), the
deposition rates of fungal spores indoors are yet to be investigated. Whilst several studies
have investigated the effect of ventilation on non-bioaerosol particle concentration
indoors (Jamriska et al., 2000; Howard-Reed et al., 2003; Wallace et al., 2004a), none of
these studies examined the effect of ventilation on the particle deposition rates of
bioaerosol particles.
In situations where the risk is high, the real-time detection and identification of
bioaerosols is becoming increasingly important. This growing need for techniques that
can be used to monitor and detect biological weapons, or to build early warning
biodetection networks, has led to an increasing focus on real-time bioaerosol
measurements (Grinshpun and Clark, 2005). In addition, real-time bioaerosol
measurements are also becoming valuable in medical and agricultural areas.
Real-time monitoring has many advantages over conventional methods, including the
short time needed for sample detection (in the order of seconds), reduced labour and
greater analytical frequency, due to the possibility of continuous monitoring. This
continuous prolonged monitoring may also increase the accuracy of the results when
compared to short-term bioaerosol collection using conventional sampling methods
(Dales et al., 1997).
The real-time instrument used in this project is the Ultraviolet Aerodynamic Particle Sizer
(UVAPS, Model 3312, TSI, St Paul, MN). It can provide real-time concentration, size
3
distribution and fluorescence measurements for particles with aerodynamic diameters of
0.5-15 µm. Fluorescence detection is performed by exciting the particles with a UV laser
beam at 355 nm and then detecting emissions at 420-575 nm.
While many investigations have been conducted to detect and measure the concentration
of bacteria using the UVAPS (Brosseau et al., 2000; Agranovski et al., 2003a;
Agranovski et al., 2003b), very limited work has been done on evaluating the method for
monitoring airborne fungi.
The aim of this project was to develop a scientific understanding of the dynamics of
fungal propagules, as well as real-time monitoring in indoor environments, for application
to fungal exposure assessments. This new knowledge of UVAPS performance with
respect to fungal spores, may also contribute to improving current real-time monitoring
techniques for measuring bioaerosols in indoor environments, as well as finding tools to
improve indoor air quality.
1.2 Overall aims of the project
In this study, the terms fungi and fungal spores refer to molds (moulds) and mold spores.
In this project, the term fungi was defined as ‘multicellular microorganisms consisting of
mycelium (a network of extensive branches bearing fruiting bodies) and reproducing
normally via asexual spore release’. Other members of the fungal kingdom, such as
yeasts, mushrooms, puffballs and rust were not included.
The overall aims of the project were:
• To investigate the dynamic behaviour of biological aerosols (fungal spores and
other fungal propagules) in indoor environments using the real-time UVAPS, and
to compare the results to those obtained for non-biological aerosols (from this
4
study and also those found in the literature), in order to develop a better scientific
understanding of bioaerosol dynamics.
• To evaluate the performance of the UVAPS in monitoring and measuring fungal
spores (viable and non-viable) under a wide range of operating conditions, and to
investigate its ability in to distinguish between fungal spores of different species
under controlled conditions.
• To compare the above results to the theoretical basis of the UVAPS design, as
well as other results found in the literature for studies that investigated bacteria
using the UVAPS, in order to provide a scientific foundation for the real-time
monitoring techniques currently used to measure bioaerosols.
1.3 Specific objectives of the study
• To evaluate the detection limit, selectivity and counting efficiency of the UVAPS,
with respect to aerosolised fungal spores.
• To correlate results obtained with the UVAPS with AGI-30 impingers.
• To develop a simple and reproducible method for the generation of fungal spores.
• To study the ability of the UVAPS to distinguish between fungal genera, under
controlled conditions.
• To investigate the effect of fungal spore age and the frequency of air current
exposure on the colony, on the fluorescent percentage of the spores.
5
• To investigate the change in spore size with age, and determine whether spore size
can be used as a co-factor to assist in distinguishing between Aspergillus niger
and Penicillium species, under controlled conditions.
• To investigate the fragmentation of fungal spores, using a wide range of variables,
such as different releasing methods and airflow velocities above fungal colonies.
• To install mechanical ventilation system in an existing chamber, in order to
simulate a typical indoor environments, for use when studying fungal spore
deposition.
• To study fungal spore release under different conditions, such as different
aerosolization methods and airflow velocities above fungal colonies.
• To investigate the deposition rate of fungal particles in indoor environments and
study the effects of ventilation on that deposition.
1.4 Account of scientific progress linking the scientific papers
This thesis contains a number of papers that have been published in, or submitted to,
refereed journals. The first paper (Chapter 3) focused on evaluating the ability of the
UVAPS to measure fungal spores. It presents the detection limit, selectivity and counting
efficiency of the UVAPS with regard to aerosolised fungal spores, and it goes on to report
the suitability of the instrument for the detection and measurement of fungal spores. In
this first study, fungal spores from Aspergillus niger (American Type Culture Collection -
ATCC 9142) and Penicillium (Australian Collection of Microorganisms - ACM 4616)
species were aerosolised under controlled conditions using the dry generation method.
Aerosols were also generated using a 6-jet Collison nebuliser. Since the UVAPS was used
6
for measuring the concentration of released fungal spores for the first time, fungal
aerosols were also simultaneously sampled using AGI-30 impinger as a reference
sampler. The UVAPS was found to be sufficiently sensitive to detect the fluorescent
biomolecules present in the fungal spores investigated, and a reasonable correlation was
found between the results from the UVAPS and the AGI-30 impinger. Another important
finding was the determination of the upper detection limit of the UVAPS for fungal
particles, which was approximately 7 × 107 particles/m3. While the Collison nebuliser
was found to be unable to generate fungal aerosols with diameters of 5 µm and larger, the
dry generation method, which was used for generating fungal spores in the subsequent
studies, was found to be simple, inexpensive, reproducible and easy to control.
The second paper (Chapter 4) focused on the ability of the UVAPS to distinguish between
two fungal strains (Aspergillus niger and Penicillium) as examples of fungal types
isolated from indoor air, under controlled conditions. The first objective was to measure
the fungal spore fluorescent percentage and fluorescent intensity for each species, as a
function of fungal age, as well as the frequency with which each fungal colony was
exposed to air currents. The second objective was to investigate the change in fungal
spore size during the three week culturing period. The study found that spore fluorescent
percentage decreased for both fungal genera as they aged, as well as with the number of
times the fungal spores were exposed to air currents. On the other hand, the fluorescence
intensity spectra were found to be valuable in discriminating between two species of the
same age, although they would not be useful when measuring spores of mixed ages, as
would normally be the case in a typical indoor or outdoor environment. The study also
showed an increase in aerodynamic diameter for the fungal spores under investigation,
over a period of time. As such, spore size distribution was also useful when
differentiating between genera of the same age, but not for genera of different ages. Based
7
on fungal spore size distributions, together with fluorescent percentages and intensities,
the study demonstrated the ability of UVAPS to discriminate between two fungal spore
genera, under controlled laboratory conditions. In the field, however, it would not be
possible to use the UVAPS to differentiate between different fungal spores due to the
presence of different micro-organisms, of varying ages, which would have been subject to
different environmental conditions. In addition, the environment may also contain non-
biological aerosols, which may fluoresce at the same wavelength as the spores do.
Submicrometer fungal fragments have more severe adverse effects than fungal spores
because they penetrate deeper in the respiratory tract and deposit into bronchi,
bronchioles and alveoli (Cho et al., 2005). Therefore, the ease with which fungal spores
fragment under different air flow rates, comparable to those found in indoor
environments, was also investigated. The third paper (Chapter 5), presents results that
show the effect of using three different aerosolization methods on the concentration of
released fungal fragments from different fungal species. The effect of different airflow
velocities above the agar plate on the concentration of released fungal fragment
propagules for each method was also investigated. The UVAPS and a Scanning Mobility
Particle Sizer (SMPS) were used to monitor the full scale of fungal spore size
distribution. In general, fragmentation was found in all three fungal spore aerosolization
methods, and it increased with increasing air flow rate. In addition, the fluorescent
percentage of fragmented samples was found to be lower than that of non-fragmented
samples, for all three methods. No fragmentation was found for flow rates similar to those
observed in typical indoor environments.
Since particle deposition rates indoors are one of the most important factors that
determine the effect of particle exposure on human health, it was also necessary to study
8
fungal particle deposition rates, in order to determine the fate of these spores and
fragmented particles in indoor environments. The fourth paper (Chapter 6), investigated
the deposition rates of fungal particles in a 20.4 m3 chamber used to simulate a typical
indoor environment. The effects of ventilation on the fungal particle deposition rates were
also investigated. The results (both with and without ventilation) were compared to the
deposition rates of non-bioaerosol particles of similar sizes, measured under the same
conditions. The study was conducted for a wide size range of particle sizes (0.54 – 6.24
µm), at three different air exchange rates (0.009, 1.75 and 2.5 h-1). It was found that the
real-time UVAPS was a useful tool for investigating the deposition of fungal particles,
however it was not found to be selective for bioaerosols, for which it was designed. The
deposition rates of the bioaerosols (Aspergillus niger and Penicillium genera) were found
to be in the same range as for non-biological particles measured under similar conditions.
The results also showed increasing deposition rates with an increasing ventilation rate, for
all particles under investigation. These results, which were reported for the first time, are
important for understanding the dynamics of fungal spores in the air.
9
References
Agranovski, V., Ristovski, Z., Hargreaves, M., Blackall, P. J. and Morawaska, L. (2003a).
Real- time measurement of bacterial aerosols with the UVAPS: performance
evaluation. Aerosol Science, 34, 301-317.
Agranovski, V., Ristovski, Z., Hargreaves, M., Blackall, P. J. and Morawska, L. (2003b).
Performance evaluation of the UVAPS: influence of physiological age of airborne
bacteria and bacterial stress. Journal of Aerosol Science, 34, 1711-1727.
Brasche, S. and Bischof, W. (2005). Daily time spent indoors in German homes -
Baseline data for the assessment of indoor exposure of German occupants.
International Journal of Hygiene and Environmental Health, 208, 247-253.
Brosseau, L. M., Vesley, D., Rice, N., Goodell, K., Nellis, M. and Hariston, P. (2000).
Differences in detected fluorescence among several bacterial species measured
with a direct-reading particle sizer and fluorescence detector. Aerosol Science and
Technology, 32, 545-558.
Byrne, M. (1998). Aerosol exposed. Chemistry in Britain, August, 23-26.
Cho, S.-H., Seo, S.-C., Schmechel, D., Grinshpun, S. A. and Reponen, T. (2005).
Aerodynamic characteristics and respiratory deposition of fungal fragments.
Atmospheric Environment, 39, 5454-5465.
Dales, R. E., Miller, D. D. and Mcmullen, E. (1997). Indoor Air Quality and Health:
Validity and Determinants of Reported Home Dampness and Moulds.
International Journal of Epidemiology, 26.
Dales, R. E., Zwanenburg, H., Burnett, R. and Franklin, C. A. (1991). Respiratory Health
Effects of Home Dampness and Molds among Canadian Children. American
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Dudney, C. S. and Copenhaver, E. D. (1984). The elements of indoor air quality. In:
Walsh, P. J., Dudney, C. S. and Copenhaver, E. D. (Eds.), Indoor Air Quality.
CRC Press, Inc., Florida.
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Fischer, G. and Dott, W. (2003). Relevance of airborne fungi and their secondary
metabolites for environmental, occupational and indoor hygiene. Archives of
Microbiology, 179, 75-82.
Grinshpun, S. A. and Clark, J. M. (2005). Measurement and characterization of
bioaerosols. Journal of Aerosol Science, 36, 553-555.
Hargreaves, M., Parappukkaran, S., Morawska, L., Hitchins, J., He, C. and Gilbert, D.
(2003). A pilot investigation into associations between indoor airborne fungal and
non-biological particle concentrations in residential houses in Brisbane, Australia.
The Science of The Total Environment, 312(1-3), 89-101.
Hobbs, P. V. (2000). Introduction to atmospheric chemistry. Cambridge University Press,
United States of America.
Howard-Reed, C., Wallace, L. A. and Emmerich, S. J. (2003). Effect of ventilation
systems and air filters on decay rates of particles by indoor sources in an occupied
townhouse. Atmospheric Environment, 37, 5295-5306.
Jamriska, M., Morawaska, L. and Clark, B. A. (2000). Effect of ventilation and filtration
on submicrometer particles in an indoor environment. Indoor Air, 10, 19-26.
Jo, W.-K. and Seo, Y.-J. (2005). Indoor and outdoor bioaerosol levels at recreation
facilities, elementary schools, and homes. Chemosphere, 61, 1570-1579.
Kim, J. L., Elfman, L., Mi, Y., Wieslander, G., Smedje, G. and Norback, D. (2007).
Indoor molds, bacteria, microbial volatile organic compounds and plasticizers in
schools – associations with asthma and respiratory symptoms in pupils.
International Journal of Indoor Environment and Health 17, 153-163.
Koskinen, O., Husman, T., Hyvärinen, A., Reponen, T. and Nevalainen, A. (1997). Two
moldy day-care centers: a Follow-up Study of respiratory symptoms and
infections. lndoor Air, 7, 262-268.
Lebowitz, M. D. (1983). Health effects of indoor pollutants. Ann. Rev. public health, 4,
203-221.
Macher, J. M. (Ed.) (1999). Bioareosols Assessment and Control. American Conference
of Governmental Industrial Hygienists, Cincinnati.
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Maroni, M., Seifert, B. and Lindvall, T. (Eds.). (1995). Indoor air quality, a
comprehensive reference book. Elsevier Science B.V., Amsterdam.
Maus, R., Goppelsroder, A. and Umhauer, H. (2001). Survival of bacteria and mold
spores in air filter media. Atmospheric Environment, 35, 105-113.
Nester, E. W., Anderson, D. G., Roberts, C. E., Nancy, J., Pearsall, N. N. and Nester, M.
T. (2004). Microbiology: A Human Perspective. (Vol. 4). McGraw-Hill, New
York.
Ott, W. R. (1982). Concepts of human exposure to air pollution. Environment
International, 7, 179-196.
Pei-Chih, W., Huey-Jen, S. and Chia-Yin, L. (2000). Characteristics of indoor and
outdoor airborne fungi at suburban and urban homes in two seasons. The Science
of The Total Environment, 253, 111-118.
Portnoy, J. M., Barnes, C. S. and Kennedy, K. (2004). Sampling for indoor fungi. Journal
of Allergy and Clinical Immunology, 113, 189-198.
Verhoeff, A. P. and Burge, H. A. (1997). Health Risk Assessment of Fungi in Home
Environments. Annals of Allergy, Asthma and Immunology, 78, 544-556.
Vijay, H. M., thaker, A. J., Banerjee, B. and Kurup, V. P. (1999). Mold allergens. In:
Lockey, R. F. and Bukantz, S. C. (Eds.), Allergens and Allergen Immunotherapy.
(2nd ed.). Marcel Dekker, Inc., New York.
Wallace, L. A., Emmerich, S. J. and Howard-Reed, C. (2004). Effect of central fans and
in-duct filters on deposition rates of ultrafine and fine particles in an occupied
townhouse. Atmospheric Environment, 38, 405-413.
Zhang, Y. (2005). Indoor air quality engineering. CRC Press LLC, Boca Raton, Florida.
12
CHAPTER 2 : LITERATURE REVIEW
2.1 Pollutants, aerosols and bioaerosols in indoor environments
Concern regarding the health effects of indoor air quality has grown, since many people
now spend most of their time indoors, in locations such as homes, offices, shopping
centres and factories (Ott, 1982; Lebowitz, 1983; Byrne, 1998; Brasche and Bischof,
2005; Bennett and Koutrakis, 2006). In industrialised countries, people often spend over
90% of their time indoors (Zhang, 2005; Bernstein et al., 2008), in environments which
are effectively isolated from outdoor influences. Indoor environments include residential,
occupational (non industrial), institutional, commercial, public, industrial and
transportation buildings (Morawska and Moore, 2000).
The pollutants found in indoor air can be inorganic, such as carbon dioxide, carbon
monoxide, nitrogen dioxide, sulphur dioxide and ozone, or organic, such as volatile
organic compounds, formaldehyde, pesticides, polynuclear aromatic hydrocarbons and
polychlorinated biphenyls (Hawthorne et al., 1984; Maroni et al., 1995; Zhang, 2005).
Pollutants can be primary or secondary pollutants (Baron and Willeke, 2001; Al-Salem
and Al-Fadhlee, 2007). While primary pollutants, such as sulfur, nitrogen, halogen,
fluorides, SO2, NO and CO are emitted directly from indoor sources, secondary pollutants
such as sulphate (SO4-2) and ozone (O3) are formed from primary compounds.
Despite a recent increase in bioaerosol research efforts, which has resulted from the
increased threat of bioterrorism and the use of biological weapons, much research still
remains to be done (Grinshpun and Clark, 2005). In general, the study of indoor
13
bioaerosols, including fungi, is aimed at discovering the cause of ongoing discomfort and
poor health, and making links between these bioaerosols and specific health effects
(Burge, 1995). It also aims to increase knowledge about their sources and their airborne
fate (Wood et al., 1993).
2.1.1 Aerosols The term aerosol refers to “an assembly of liquid or solid particles suspended in a gaseous
medium long enough to enable observation or measurement” (Willeke and Baron, 1993).
Aerosols can originate from many sources, with each source generating different aerosols.
These sources can be divided into two major classes: natural sources, such as volcanoes,
ocean spray, fungi and pollen; and anthropogenic sources, which include industrial,
agricultural, commercial and transportation (Zannetti, 1990; Baron and Willeke, 2001).
The sources of indoor aerosols can include tobacco smoke, combustion products from gas
or other fuels (such as residential wood burning or the combustion of diesel vehicle
fuels), and emissions from building materials, as well as household cleaning products
(Calle and Zeighami, 1984; Moschandreas, 1984; Willeke and Baron, 1993; Maricq,
2007). Other examples of aerosols include soot, silicon, sodium, titanium and vanadium,
as well as physical pollutants such as particulate matter, asbestos, mineral fibres and
radon (Dudney and Copenhaver, 1984; Glantz, 1984; Zannetti, 1990; Maroni et al., 1995;
Zhang, 2005; Pastuszka, 2009). In general, the size of aerosol particles ranges from 0.001
- 100 µm and their size often relates to their method of generation. While combustion
leads to small particles, mechanical generation methods form relatively large particles
(Baron and Willeke, 2001). The most widely used definition of particle size is
‘aerodynamic equivalent diameter’ which can be defined as “the diameter of a spherical
particle of unit density which has the same settling velocity as the particle of interest”
(Knutson and Lioy, 1989).
14
Aerosols can be classified in different ways, either by their generation source (whether
mechanical or another process) or by particle size (the most common classification
method). In terms of size, particles are generally classified as being either PM2.5 or PM10
(Spurny, 1999b; Matti Maricq, 2007), where PM2.5 and PM10 refer to the mass
concentration of particles with aerodynamic diameters smaller than 2.5 µm and 10 µm,
respectively.
2.1.2 Bioaerosols While Macher (1999) defined biological aerosols as those airborne particles that are
living or originated from living organism, Cox and Wathes, (1995) defined them as “an
aerosol of biological origin which exerts a biological action in animals and plants by
virtue of its viability, infectivity, allergenicity, toxicity, pharmacological, or other
biological properties, with an aerodynamic diameter in the range 0.5 - 100 µm”. Viruses,
which may also be considered bioaerosols (Grinshpun and Clark, 2005), are in the size
range of 0.02 - 0.3 µm (Willeke and Baron, 1993). Others list a much broader size range
for biological aerosols (Macher, 1999; Grinshpun and Clark, 2005). Grinshpun and Clark
(2005) reported bioaerosols within the range ~ 0.001 µm to ~100 µm, while and Macher
(1999) reported them from <0.01 µm to particles greater than 100 µm. Bioaerosols
include pollen, fungi, fungal spores, bacteria, viruses, domestic dust mites and their
excreta, alga, protozoa, toxins, fragments, volatile organic compounds and the particulate
waste products of animals, plants and other microorganisms (Dudney and Copenhaver,
1984; Maroni et al., 1995; Muilenberg, 1995; Macher, 1999; Hobbs, 2000; Grinshpun and
Clark, 2005; Zhang, 2005).
2.1.2.1 Concentration of fungal spores in indoor and outdoor environments
The concentration and characteristics of particular pollutants in indoor environments
depend on two factors: the penetration of airborne pollutants from the outdoor
15
environment, and the internal generation of these pollutants (Maroni et al., 1995).
Outdoor microorganisms primarily grow in moist soil, on the surface of leaves and in
plant litter (Shaughnessy et al., 1999). While bioaerosols, including pollen, bacteria, algae
and insect fragments, are present in outdoor air, fungal spores tend to dominate in indoor
air (Muilenberg, 1995). These aerosols reach indoor environments through natural or
mechanical ventilation, windows, doors, cracks in the buildings walls or they may be
transported indoors by people (on their shoes, clothes or skin) and animals (Muilenberg,
1995). Penetration efficiency is the fraction of outdoor particles that penetrate indoors,
and it has been found to be roughly the same for both fine and coarse particles (Wallace,
1996). The concentration of indoor particles is highly variable and house-specific.
Outdoor particle concentrations are strongly affected by outdoor particle emission sources
and meteorological parameters. Usually the outdoor fungal spore concentration is 103 -
104 spores/m3, but sometimes it can reach as high as 106 - 109 spores/m3, as has been
observed during crop harvesting (Lacey and Dutkiewicz, 1994). In general, the highest
number of outdoor fungal spores is found during summer and autumn (Dix and Webster,
1995), and during these seasons, the main source of fungi in indoor air is from outdoor
sources (Maroni et al., 1995).
In general, little information is available on the background concentration levels of
biological agents in indoor environments, and significant spatial, temporal and seasonal
variations have been reported in the literature (Lis and Pastuszka, 1997). With respect to
fungal spores, many factors affect their concentration in indoor air. These factors include
outdoor concentration, moisture, type and operation of the ventilation system, seasonality
and house characteristics (Lawton et al., 1998; Hargreaves and Parappukkaran, 2000).
16
A Brisbane study showed that fungal spore counts tend to be higher in the 2-10 µm range
and are very high in Brisbane during the winter months, from the end of April to mid-
June (Glikson et al., 1995). Hargreaves et al. (2003a) reported that the concentration of
airborne fungi in 14 residential suburban houses in Brisbane was 810 ± 389 CFU/m3,
while Lee et al. (2006) reported that the concentration of airborne fungi in 6 Cincinnati
homes was typically between 0 - 1362 CFU/m3. Hyvärinen (2000) reported the
concentration range of viable fungi in indoor environments in Finland was in the range
101-105 CFU/m3, while airborne spore concentrations were found to range from 103-104
CFU/m3 in a heavily colonised crawl space in Finland (Kurnitski and Pasanen, 2000).
In comparison to non-biological concentrations, the concentration of biological particles
(bacteria or fungi) in typical indoor environments was generally found to be significantly
lower, with concentrations of biological viable particles in the order 101-104 /m3 and non-
biological particles in the order of 109-1011/m3 and 106-107/m3 for submicrometer and
supermicrometer particles, respectively (Morawska and Salthammer, 2003b).
2.1.3 Fungi
2.1.3.1 Definition and taxonomy
In Whittaker’s five-kingdom system, “Fungi” are said to have the following defining
characteristics: multicellular organism (except for yeasts, which are unicellular), non-
photosynthetic, nutrient absorpting, chemoheterotrophic (using chemical compounds as
energy sources and organic compound as carbon sources (Moor-Landecker, 1996; Perry
and Staley, 1997), and reproducing via sexual and asexual means. The more recent and
widely accepted phylogenetic classification system by Woese defines fungi as one of the
kingdoms in the domain “Eukarya”. The kingdom of fungi includes the macroscopic
17
fungi, such as mushrooms, bracket fungi and puffballs, and microscopic fungi such as
molds, yeasts, dermatophytes and rusts.
Fungi are eukaryotic organisms that belong to a kingdom different from plants and
animals (Levetin, 1995). The major difference between eukaryotic microorganism cell
and the prokaryotic microorganism cell (bacteria) is that the nucleus of eukaryotic cell is
bound by a membrane, while the nuclear material in the prokaryotic cell is not bound by a
membrane but is in direct contact with the cytoplasm (Perry and Staley, 1997).
Mycologists use sexual lifecycle stages of fungi (teleomorphic) to classify them, while
the asexual stages (anamorphic) are classified separately. Based on their stages of sexual
reproduction, the fungi found in indoor environments can be placed into one of the
following groups: Zygomycetes, Ascomycetes and Basidiomycetes, while fungi with
asexual stages only (imperfect fungi or Deuteromycetes), can be placed in Blastomycetes,
Hypomycetes or Coelomycetes groups (Burge and Otten, 1999)
It is estimated that there are at least one million different fungal genera (Vijay et al.,
1999b), and possibly up to 1.5 million according to Hawksworth (Dix and Webster,
1995). Fungi are predominantly terrestrial, however many aquatic fungal species are also
being found, the majority of which are found in fresh water (Ingold, 1979; Dix and
Webster, 1995; Nester et al., 2004).
Fungi can be grouped in three different categories: saprophytes, which get their food from
non-living organic matter (indoor molds tend to be saprophytes, due to their ability to
grow on inanimate surfaces); decomposers, which decay dead organic organisms; and
parasites, which obtain their nutrients from the tissues of a living host (Levetin, 1995).
18
2.1.3.2 Structure
The vast majority of fungi are composed of hyphae, thread-like tubular structures,
bounded by walls, and forming extensive branched, often anastomosing systems called
mycelia (Griffin, 1994). Mycelia have a very high surface to volume ratio and hence, they
exploit a large surface area of the substratum, absorbing nutrients with a minimum
expenditure of biomass (Dix and Webster, 1995). A colony is made up of a visible mass
of interwoven hypha that form mycelium. they may appear cottony, velvety, granular or
leathery, and in a variety of colours, including white, grey, black, orange, green or yellow
(Burge and Otten, 1999). The cottony or other appearance mycelium is due to the
formation of aerial hyphae whose purpose is solely to disseminate asexual spores. The
most common mycelial fungi found in the indoor environment are molds (moulds) (Burge
and Otten, 1999), an example of which is the white mass that can be seen on moldy bread
(Nester et al., 2004). The hyphae are mostly septate, divided by cross-walls into distinct
cells, with a nucleus for each cell. However, some are not septate and can have many
nuclei in the same thread (Smith, 1976). In both cases, hyphae can transfer the nutrients
from the rich sites within the mycelium to the other places where the same nutrients are in
short supply. This happens because the septa in the hypha do not divide it completely, but
form partial divisions, allowing the movement of water, solutes, organelles and even
nuclei (Moore, 2003).
2.1.3.3 Fungal spores: size, shape and density
Fungal spores can contain as little as one or many cells of differing size, colour, shape
and method of formation (Levetin, 1995). There is no exact size for fungal spores, but
studies show that they range from less than 2 µm to more than 100 µm in size (Levetin,
1995). The spores of most fungal species are found within the range 2-20 µm (Vijay et
al., 1999b). However, in many studies, the mold found in buildings are often smaller than
19
10 µm (Reponen et al., 1994). Reponen et al. (1994) also found that the geometric mean
diameters of fungal spores in buildings with mold problems range from 1.1-5.7 µm in
indoor air and 2.1-4.2 µm in outdoor air.
Fungal spore density is generally around 1.1 g/cm3 (Griffin, 1994; Grinshpun et al.,
2005), although Sawyer et al. (1994) found that the conidial densities of Zoophtora
radicans, Conidiobolus obscurus, and Conidiobolus thromboides (spore densities were
determined from the refractive index of spores measured by interference microscopy)
were slightly lower, with values of 1.10 g/cm3, 1.06 g/cm3 and 1.06 g/cm3, respectively.
Most fungal spores vary in the number of cells in a chain, thickness of cell wall (in
general they have thick walls to survive extremely severe conditions) and the way they
attach to each other and to their conidiophores. Spore size and shape are widely used
characteristics in fungal taxonomy and can often be used to identify genera (Parmasto and
Parmasto, 1987). The characteristics of some of the most common fungal species found
both indoors and outdoors (i.e. Penicillium, Aspergillus and Cladosporium) (Walsh et al.,
1984; Reponen et al., 1994; Verhoeff et al., 1994; West, 1998; Rankovic´, 2005; Rabito et
al., 2008) are presented below:
While Penicillium sp. spores have a greenish blue colour with a subglobose shape and
range from 2-4 µm in size (Ramirez, 1982), Aspergillus niger spores have a brownish
black colour with a globose to subglobose shape and range from 2.5-5.0 µm in size
(Raper et al., 1965). C. cladosporioides spores have an ellipsoidal to limoniform shape
and range from 3.0-7.0 (-11) x 2.0-4 (-5.0) µm in size (Samson and Hoekstra, 1994).
They are dark olivaceous to black in colour, have a velvety texture and are often slow
growing (Tamsikar et al., 2006).
20
2.1.3.4 Growth and reproduction
Fungi reproduce through the production of spores, which can be either sexual spores,
asexual spores, or both, when the conditions (food, temperature, humidity, and pH) are
suitable. Fungal spores are considered to be both the beginning and the end of the fungal
development cycle (Smith, 1975) and they differ from gametes in that they have the
ability to develop into a new individual without requiring fusion with another
reproductive cell (Damare et al., 2008). Most molds are asexual forms of the Ascomycota
and the asexual spores produced are referred to as “conidia” which means “dust”. This
refers to the way that the spores freely disperse into the atmosphere and float around like
dust.
Water, carbon sources and oxygen are universally required to activate spore germination
(Damare et al., 2008). Under adverse environmental conditions, some fungi form thick-
walled dormant spores (chlamydospores) which form from transformed vegetative
hyphae (Levetin, 1995). These dormant spores are metabolically inactive and are not
usually dispersed (Griffin, 1994; Levetin, 1995). Fungi can remain dormant for several
years (Raghukumar et al., 2004) and can develop into a vegetative form where suitable
growth conditions are present (Damare et al., 2008).
When fungal dormant spores are placed in a suitable growth environment, the
germination process starts off with the spore gradually swelling, so that its diameter and
biomass both increase, after which the hyphal element (germ tube) then extends from the
enlarged spore (Bosch et al., 1995). While Damare et al. (2008) regard the spore to have
germinated if the germ tube is at least as long as the width of the spore, Bosch et al.(1995)
used a germ tube of a length of at least 1.5 times the diameter of the examined conidia to
define germination. As the germ tube (hypha) grows, extending in length as well as
21
branching, it forms a network called ‘mycelium’, with a typical hypha diameter around 5-
6 µm (Dix and Webster, 1995; Gow and Gadd, 1995).
Fungi can grow on virtually any substrate including glass, paint, textiles and electrical
equipment (Denizel, 1974; Hargreaves and Parappukkaran, 2000) and they often produce
a very large quantity of spores in a very short time. Fungi can be found both in indoor air,
as well as on surfaces such as floors, walls, ceilings, furniture and in the Heating,
Ventilation and Air Conditioning system (HVAC) (Shaughnessy et al., 1999). Fungi can
also be found in grain, damp basements, bathrooms, refrigerator drip trays, water-
damaged carpets etc.
Although high and low temperature, dryness, low pH and antifungals can inhibit fungal
growth, fungi can tolerate and survive many of the extremes of environmental exposure.
As such, it is not surprising to find fungi virtually everywhere, from the peaks of
mountains (Dubey et al., 1994; Cripps, 2001) to the deep sea and even in the arctic
(Muilenberg, 1995; Raghukumar et al., 2004; Damare et al., 2006). For example,
culturable fungi were recovered from a deep-sea sediment core obtained from a depth of
5904 m in the Chagos trench in the Indian Ocean (Raghukumar et al., 2004). Damare et
al. (2006) also reported the occurrence of fungi in deep-sea sediments from a depth of
5000 m in the Central Indian Basin, where a total of 181 cultures of fungi (dominanted by
Aspergillus species) were isolated by a variety of isolation techniques.
Many environmental factors, such as nutrition, temperature, relative humidity, light,
pressure and carbon dioxide influence fungal sporulation (Griffin, 1994; Ren et al., 2001).
While fungi can live in dark, low-oxygen niches, they do demonstrate a relationship with
oxygen and light (Jennings and Lysek, 1996). Light affects fungal sporulation rather than
mycelial growth. For example, if the fungus is growing in a closet and the light is left on,
22
the air becomes warmed and the moisture level falls, which then serves to prevent further
fungal growth (Burge and Otten, 1999). On the other hand, some fungi require a very
specific cycle of light and dark, and even a certain wavelength of light before sporulation
can occur (Burge, 1995). The temperature range for mycelia to grow and reproduce
ranges from 0-40 oC, but the majority of indoor fungi grow optimally between 10-35 oC
(Maroni et al., 1995). For example, Sautour et al. (2001) found that the optimum growth
temperature for Penicillium chrysogenum, Aspergillus flavus, Cladosporium
cladosporioides and Alternaria alternata species on Potato Dextrose Agar were 25oC,
31oC, 26oC and 25oC, respectively. In a study by Ren et al. (2001), they found that the
spores of several deep-sea Aspergillus isolates did not germinate at temperatures around
4-5 oC (the ambient temperature of the deep sea). Whilst fungi can grow at a pH ranging
from 2.2 to 9.6, they prefer a pH of 5.0 or lower (Nester et al., 2004). Fungi can also
germinate under high hydrostatic pressure (Ren et al., 2001). For example, Ren et al.
(2001) found that the spores of several deep-sea Aspergillus isolates germinated under an
elevated hydrostatic pressure at 30 oC, and that the percentage of germination decreased
gradually with increasing pressure.
2.1.3.5 Fungi and moisture
Moisture supports biological growth (Macher, 1999). Water activity is a measure of the
water in a substrate which an organism can use to help it grow (Griffin, 1994; Burge and
Otten, 1999) and it is directly related to the relative humidity of the substrate (i.e. if the
substrate relative humidity is 80%, the water activity is 0.8) (Levetin, 1995).
Many fungi and yeast can grow at a water activity (aw) of 0.7 (Godish, 1989; Levetin,
1995; Nester et al., 2004), however the minimum water activity needed for fungal growth
on building substrates is 0.75 (Grant et al., 1989 ). The minimum water activity and the
23
optimal water activity needed for fungal species differ from one species to other. For
example, Sautour et al. (2001) found that the minimum water activity required for
Penicillium chrysogenum, Aspergillus flavus, Cladosporium cladosporioides and
Alternaria alternate growth were 0.810, 0.822, 0.850 and 0.883, respectively, while the
optimal water activity was 0.985, 0.974, 0.983 and 0.987, respectively. It has also been
reported that some mesophilic fungi (which grow best at a moderate temperature) require
a particularly high water activity, while xerophilic fungi (which can survive in dry
conditions) require a much lower water activity (Wood et al., 1993).
Water damage and moldy odours are often associated with elevated levels of indoor fungi
(Dales et al., 1997). It has been found that the source of most indoor air contaminants is
the microbiological growth often found in the moist sections of a building HVAC system,
including drip pans and humidifiers (Maroni et al., 1995; West, 1998). A number of
epidemiological studies conducted in the U.K., U.S.A. and Canada and other places
suggest that exposure to dampness and mold in homes is strongly associated with a
number of respiratory symptoms (Dales et al., 1991; Miller, 1992). In susceptible
individuals, the exposure to moisture-associated microbes may increase the risk of
autoimmune disease (Husman, 2004).
2.1.3.6 Fungal fragmentation
In general, submicrometer fungal fragments cause more severe adverse effects than
fungal spores because they can penetrate deeper in the respiratory tract and deposit in the
alveolar region of the lungs (Cho et al., 2005). The occurrence of fragments in the air has
been confirmed by many studies (Sorenson et al., 1987; Li and Kendrick, 1995) and the
contribution of fungal fragment contribution to overall exposure is very high (Reponen et
al., 2007). Wild-type fungi collected indoors showed that airborne hyphae and fragmented
24
conidia were potential airborne allergens (Green et al., 2005), while some fungal
fragments have also been found to contain mycotoxins (Brasel et al., 2005).
Many studies have investigated the potential release and quantitative measurement of
fungal propagules (spores, conidia, hyphae, mycelia etc) (Górny et al., 2001; Górny et al.,
2003; Górny, 2004; Cho et al., 2005). While Cho et al. (2005) studied the aerodynamic
characteristics of the released fungal fragments, others investigated the effects of many
variables, such as air velocity over colonies, fungal species, vibration, humidity and
colony structure, on the release of fungal fragments (Górny et al., 2001; Górny et al.,
2003; Górny, 2004).
Górny et al., (2003) reported that the release of Streptomyces albus fragments increased
14-fold when the air velocity across the agar surface increased from 0.3 m/s to 29.1 m/s,
while Górny et al., (2002) found that the increase in fragments also depended on colony
growth surface. Górny et al., (2002) also found that fragments from Aspergillus
versicolor and Penicillium melinii species share common antigens with their spores,
which makes it possible to confirm the fungal origin of the fragments. These fragments
were either; mycelium, aerosolised from microbiologically contaminated surfaces; pieces
of spores and fruiting bodies, or they may have formed through the nucleation of some
secondary metabolites of the fungi, such as volatile organic compounds (VOCs) (Górny,
2004).
2.1.3.7 Fungal secondary products
When fungi compete for the available humidity, temperature and nutrients, the
microorganism can release toxic and antibiotic metabolic by-products to kill any
competitors (Macher, 1999). Thousands of the secondary products (agents) which are
produced by fungi have been chemically characterised (Levetin, 1995) and they include
25
mycotoxins, antibiotic, alkaloids, VOCs and structural components such as β-(1→3)-D-
glucans (Burnett, 1976; Levetin, 1995; Burge and Ammann, 1999; Macher, 1999).
Mycotoxins are non-volatile, relatively low molecular-weight products, while antibiotics
are normally large molecules, which have also been recognised as a valuable substance in
contemporary medicine (Burge and Ammann, 1999). Fungal toxins can be classified into
mushroom toxins (or poisons) and mycotoxins, which is formed by the hyphae of
common molds (Levetin, 1995). There are hundreds of different mycotoxins and the most
familar of them are produced by the fungi growing on grain and nuts (Levetin, 1995).
When the concentration of fungal materials, such as hyphae, spores and fragments of
substrates is high, there is also a higher possibility that mycotoxins will be present
(Muilenberg, 1995).
Common fungal mycotoxins include aflatoxins, Ochratoxin A, Patulin and Griseofulvin,
which are produced by Aspergillus flavus, Aspergillus Ochraceus, Penicillium expansum
and Penicillum griseofulvin, respectively (Burge and Ammann, 1999). The type and
amount of mycotoxin produced depends on the fungal strain and the substrate used, and a
mycotoxin effect is often specific to particular target organs. For example, aflatoxins
target the liver, while ochratoxins target the kidneys (Lacey and Dutkiewicz, 1994).
When ingested, the mycotoxins produced by fungi can be toxic, carcinogenic, teratogenic
or mutagenic to both man and animals (Lacey and Dutkiewicz, 1994; Burge and
Ammann, 1999). In contrast, glucans are glucose polymers that are found in most fungal
cells and β-(1→3)-D-glucans act as potent T-cell adjuvants, which essentially act as anti-
tumor agents (Burge and Ammann, 1999).
Many fungi produce allergens (Macher, 1999) and the allergen content can vary with
culture age, type of substrate and each strain within a species. Allergens are biological or
26
chemical substances that cause an immune response, called an allergic reaction. Most
allergens are large molecules, with a molecular weight between 10,000-80,000 Da
(Levetin, 1995). The biological sources of some common airborne indoor allergens are
fungi and fungal spores, bacteria, house dust mite, cockroaches, birds, cats, rodents and
dogs (Rose, 1999). Allergens may be found in the form of aerosolised spores, excretion
from microorganisms and/or fungal fragments (Górny et al., 2002). While antigens are
compounds that are recognised by antibodies of any class, allergens are more explicit in
that they trigger a specific immunoglobin class E or G allergic response (Levetin, 1995).
Antigens cause diseases such as allergic asthma, allergic rhinitis, allergic mycosis and
hypersensitivity pneumonitis (Rose, 1999).
Spore germination was found to lead to a significant increase in the percentage of spores
eluting a detectable allergen (Green et al., 2003). Green et al. (2003) found that all of the
spores that germinated from 11 fungal species displayed allergen elution from their
hyphae, and 8 of the 11 species showed a significant increase in the percentage of spores
eluting detectable allergen.
As part of their metabolism, various species of fungi also produce microbial volatile
organic compounds (MVOCs). These compounds are responsible for the typical ‘moldy’
scent associated with many fungi (Levetin, 1995; Maroni et al., 1995; Fink-Gremmels,
2008) (Harris et al., 1986). The MVOCs produced by fungi include alcohols, aldehydes,
ketones, aromatics, amines, terpenes, chlorinated hydrocarbons and sulphur containing
compounds (Batterman, 1995). The prominent compounds among them are short-chained
alcohols and aldehydes (Levetin, 1995) and the specific MVOCs which have been
identified in numerous studies include 3-methyl-1-butanol, 3-octanol or 1-octan-3-ol, 3-
octanon and 1-octen-3-ol (Batterman, 1995). These MVOCs may be responsible for
27
symptoms such as headaches, dizziness and eye and mucous membrane irritation, which
are often associated with indoor fungal contamination (Levetin, 1995).
2.1.3.8 Microbial growth prevention
The prevention of indoor microbial growth is possible if the factors that allowed its
growth are identified and controlled. For example, biological growth depends on an
adequate water supply and hence, controlling moisture sources in a building may assist in
the prevention or limitation of microbial growth (Shaughnessy et al., 1999). However,
because humidity is a primary physical factor that affects thermal comfort (Soebarto et
al., 2004), it is also important to maintain humidity at a level that ensures occupant
comfort (i.e. between 30 and 70%) (Bearg, 1993).
If prevention fails, the physical removal of microbial growth from porous and non-porous
materials is very important (Shaughnessy and Morey, 1999; Shaughnessy et al., 1999). In
general, biocide and antimicrobial agents are used to remove the microbial growth in
HVAC systems and on building materials or furniture (Cole and Foarde, 1999). While
biocides are chemical or physical agents capable of killing or inactivating
microorganisms, including vegetative fungi and fungal spores, antimicrobial agents are
compounds capable of suppressing microbial growth (Cole and Foarde, 1999). Microbial
growth that has been treated with biocides should be still removed because their antigenic
and toxic properties are not neutralised by the biocide (Cole and Foarde, 1999). The most
common biocide classes are alcohols, aldehydes, halogens, phenolic compounds,
hydrogen peroxide and quaternary ammonium compounds, while the most common
antimicrobial agents are phosphated quaternary amine complex and tri-n-butyltin maleate
(Cole and Foarde, 1999).
28
Physical disinfection is another method used for killing and/or removing pathogenic
microorganisms, however there is often no quantitative standard associated with these
methods (Cole and Foarde, 1999). Physical disinfection methods include germicidal
ultraviolent radiation, ionisation and thermal radiation (Cole and Foarde, 1999; Nardell
and Macher, 1999). In addition, mycelial inhibitors, such as Chenopodium ambrosioides
Linn. (Chenopodiaceae) oil has also been used (Kumar et al., 2007). The oil exhibits a
broad fungitoxic spectrum against Aspergillus niger, Aspergillus fumigatus,
Botryodiplodia theobromae, Fusarium oxysporum, Sclerotium rolfsii, Macrophomina
phaseolina, Cladosporium cladosporioides, Helminthosporium oryzae and Pythium
debaryanum at 100 μg/ml (Kumar et al., 2007).
2.1.3.9 Benefits and impacts of fungi
2.1.3.9.1 Benefits
Microorganisms, including fungi, have been used to produce foods such as yoghurt,
cheese, pickled vegetables and other fermented products. Fungi also play an important
role in the recycling of minerals and carbon in the biosphere, by feeding on organic
debris, lignocellulose, plant litter and waste (Moore, 2003; Nester et al., 2004; Dritsa et
al., 2007; Zhang et al., 2008). Some of the common genera of soil fungi involved in
nutrient cycling, including Penicillium and Aspergillus (Maier et al., 2000) have the
ability to produce many compounds that are used in the production of materials important
to the food, drug and chemical industries (Griffin, 1994; Perry and Staley, 1997). For
example, Aspergillus niger produces citric acid as a by-product of fermentation, which is
used as a preservative (Ingold, 1979; Brooke, 1994), while Penicillium notatum produces
penicillin, which is used as a medical antibiotic (Ingold, 1979). Microbial, including
fungal, biotechnology is also used in the production of steroid drugs and hormones. For
example, a variety of steroid drugs, such as anti-inflammatories, diuretics, anabolic
29
steroids, the contraceptive pill, anti-androgenic steroids, progestational and anticancer
agents all utilise microbes in their production (Mahato and Garai, 1997; Zaks and Dodds,
1997; Kristan et al., 2007).
2.1.3.9.2 Impacts
Impact on Human Health
In general, substantial epidemiological evidence suggests that fine airborne particulate
matter (PM) has an adverse effect on human health (Pope et al., 2004). For example,
Atkinson et al. (2001) confirmed that airborne particle concentrations are positively
associated with increased numbers of hospital admissions for respiratory diseases in
Europe, while in six US cities, a stronger association was found between mortality and
fine airborne PM than with coarse airborne PM (Schwartz et al., 1996).
There is increasing scientific and medical evidence to suggest that exposure to biological
aerosols has significant implications for a person’s health. Diseases like tuberculosis,
diphtheria and legionellosis are caused by airborne infectious microorganisms (albeit
bacteria) which have penetrated and deposited into the respiratory tract (Maus et al.,
2001).. For example, fungi have been found in the pulmonary parenchymal tissue
(Cordasco et al., 1957), and Scedosporium apiospermum has also been found in the
human lung (Zaas, 2002). In addition, Coccidioides immitis can cause highly infectious
respiratory diseases such as coccidioidomycosis (Larone, 2002).
Fungal infections also cause major complications in cancer and leukemia patients
(Martino and Girmenia, 2000). Since immuno-compromised patients (as a result of
immunosuppressive drugs and/or an immune deficiency disease) often do not produce a
pronounced cellular response to fungal infections, mold infections, which rarely impact
on healthy individuals, may be fatal for those suffering immunodeficiency, or recovering
30
from burns or surgery (Kowalski, 2000), (Larone, 2002). For example, pathogens such as
Aspergillus fumigatus are mostly fatal when they invade immuno-compromised patients
(Maroni et al., 1995). McNeil et al.(2001) reported that death due to mycoses infectious
disease increased from the tenth most common cause of death in the United States in
1980 to the seventh most common cause in 1997, and that these deaths occurred most
commonly in individuals with compromised immune systems, such as patients with AIDS
or malignancy.
The fungal spores of pathogenic species may lose their infectivity when subjected to
harsh conditions, however most allergenic spores retain their allergenic properties even
when the spore loses its viability (Godish, 1989; Levetin, 1995). Fungal diseases are
generally not transmitted from person to person (Steenbergen and Casadevall, 2006).
Molds are responsible for many allergic diseases in humans, including hay fever, asthma,
allergic bronchopulmonary mycoses and hypersensitivity pneumonitis (Pons and Belaval,
1961; Lacey and Dutkiewicz, 1994; Macher, 1999; Vijay et al., 1999b; Nester et al.,
2004; Kim et al., 2007; Rabito et al., 2008). It is also highly likely that the toxigenic
properties of fungi, together with the allergenic reaction it invokes, are involved in mold-
induced asthma (Maroni et al., 1995). Mycotoxins have also been found to result in health
risks for humans and animals (Maroni et al., 1995; Fink-Gremmels, 2008).
The fungal species under investigation in this study (Aspergillus, Penicillum and
Cladosporium) all may cause human infections. Aspergillus species have the ability to
colonise the human respiratory tract and the potential to act as a powerful allergen
resulting in Aspergillus asthma and allergic bronchopulmonary aspergillosis (Tomee and
van der Werf, 2001). Approximately 175 species of Aspergillus are known, but only
about 20 have been found to cause diseases. For example, Aspergillus fumigatus,
31
Aspergillus flavus, Aspergillus niger and other Aspergillus spp. have been known to cause
Aspergillosis (Chambon-Pautas et al., 2001; Larone, 2002; Falvey and Streifel, 2007).
Penicillium spp, on the other hand, has been known to cause cornel, cutaneous, external
ear, respiratory and urinary tract infections (Larone, 2002). Of the 500 described species
of Cladosporium, only two are known to be truly pathogenic to humans and animals, C.
carrionii and C. bantianum. C. cladosporioides has also been implicated as a pathogen in
some unusual circumstances. For example, one report states that it was the cause of
phaeohyphomycosis in the sebaceous cysts of one patient (Tamsikar et al., 2006).
As the majority of fungal spores (including (Aspergillus, Pencillum and Cladosporium)
are small enough to penetrate into the lower regions of the lung, they often cause allergic
reactions and hypersensitivity (Cuijpers et al., 1995; Verhoeff and Burge, 1997b; Nester
et al., 2004)
Impact on Animals, Plants and Building Materials
Fungi have a large impact on animals and plants (Cooke, 1977; Maier et al., 2000; CAST,
2003; Steenbergen and Casadevall, 2006), crops (Pitt et al., 1994) and stored seeds
(Christensen, 1965; Nester et al., 2004). Approximately 300 out of more than 100,000
fungal species have a pathogenic impact on animals and plants (McNeil et al., 2001). The
fatal disease cutaneous chytridiomycosis, which is caused by a member of the phylum
Chytridiomycota, was believed to be behind the marked decline of montane riparian
amphibian populations in Queensland (Australia) and in Central America (Berger et al.,
1998). Fungal plant diseases also have a significant economic impact. For example, in
1993 leaf rust (a type of wheat rust) was responsible for the loss of over 40 million
bushels of wheat in Kansas and Nebraska alone (Maier et al., 2000). Fungi can also
damage buildings, making them unpleasant to occupy (in terms of both smell and
32
appearance) (Portnoy et al., 2004), as well as attacking clothes, leather, wood and many
other materials.
2.1.4 Summary The concentration and characteristics of aerosols and bioaerosols in the indoor
environment depend on two factors: the penetration of airborne particles from outdoors;
and the internal generation of these pollutants.
Since people spend most of their time indoors, concern regarding the health effects of
indoor air quality has grown significantly in recent decades. A large number of studies
reported that exposure to biological aerosols (including fungi) had significant health
implications for humans and that fungi also have large impact on animals and plants.
Fungi are multicellular, chemoheterotrophic organisms (except for yeasts, which are
unicellular), that absorb nutrients and reproduce via sexual and asexual spores. The most
common mycelial fungi found in the indoor environment are molds.
Fungal spores contain as few as one and up to many cells, which differ in size, colour,
shape and method of formation. The spores of most fungal species range from 2-20 µm in
size and fungal sporulation is affected by many environmental factors such as nutrition,
relative humidity and temperature.
When competing for available resources, such as humidity and nutrients, microorganisms
produce toxic and antibiotic metabolic by-products called secondary products. These
secondary products of metabolism include mycotoxins, antibiotics, alkaloids and VOCs.
The occurrence of fungal fragments in the air has been confirmed by many studies.
Submicrometer fragments have more significant adverse effects than fungal spores,
33
because they penetrate deeper into the respiratory tract and deposit into the alveolar
region of the lung.
The prevention of indoor microbial growth is possible if the factors that facilitate growth
(especially moisture) are controlled. Biocide and antimicrobial agents may also play a
role in removing the microbial growth in indoor environments.
In regard to fungal fragmentation, it was discovered that there are still many gaps in
knowledge which need to be addressed, such as:
1. While previous studies have investigated the effect of air velocity on fungal fragment
release, they did not use a sufficiently wide range of air velocities, similar to those that
are produced by natural or mechanical ventilation in buildings.
2. Previous studies have not investigated the effect of different fungal aerosolization
methods on fungal fragment release.
3. The mechanisms of fungal fragmentation remain unclear.
4. The part (parts) of fungi which represents the source of fungal fragments found in
indoor environment is yet to be identified.
34
2.2 Fungal spore dynamics in indoor air
2.2.1 General Introduction It is expected that the dynamics of particles in the air will be governed, to a large extent,
by the physical characteristics of the particles, of which size is one of the most important.
Assuming they are governed by the same physical laws as non-biological particles of the
same particle size, the dynamics of fungal spores can also be determined according to
their aerodynamic behaviour. If, however, their biological properties also play a role, then
biological properties will also play a part in determining their airborne dynamics.
It is widely accepted that biological and non-biological particles of similar sizes will
display similar dynamics in air, responding to the action of the same forces (Baron and
Willeke, 2001). However, further experimental investigations are needed to support this
assumption. The types of physico-chemical processes that could be of importance include
coagulation, sedimentation and diffusional deposition, as well as dispersion, transport,
and penetration through ventilation and filtration systems.
The dynamic processes that control particle transport are significantly affected by particle
size. While convection, Brownian diffusion and electrophoresis are important for particles
< 0.1 µm in diameter, thermophoresis is important for particles < 1 µm in diameter.
Inertial effects and sedimentation are also important for particles of diameters > 1 µm
(Chen and Chan, 2008).
If the dynamics of fungal spore particles in indoor air are expected to be governed, to a
large extent, by the particle physical characteristics, then fungal transport and deposition
can be expressed as follows:
35
Fungal spores range in size from 0.5 - 100 µm, while their fragments are less than 0.5
µm. Those particles < 0.1 µm are governed by Brownian motion (Chen and Chan, 2008)
and their displacement may be expressed by a simplified form of the Einstein equation
(Cox and Wathes, 1995):
)rt( 10 5 X 6-×=
Where X = root mean square of particle displacement
t = time (sec)
r = particle radius (cm)
For fungal spores with diameter >1 µm, gravitational force is much more important than
diffusion (due Brownian motion). Here, according to Stokes law, when the drag frictional
and the gravitational forces are equal, the terminal velocity for falling of a spherical
particle is (Baron and Willeke, 2001):
η
ρ 18
Cgd v2
=
Where v = terminal velocity (cm/s)
ρ = particle density (g/cm3)
d = particle diameter (cm)
η = air viscosity (g/cm s)
C = Cunningham slip correction
36
2.2.1.1 Ventilation and its effects on fungal spore deposition rates
After the worldwide energy crisis in the mid-1970s, and in an effort to minimise the loss
of energy through the building envelope, the buildings in western developed countries
were built with more airtight and better insulated materials. The synthetic materials and
chemical products used in these airtight buildings, together with the low ventilation rate,
has resulted in elevated concentrations of volatile organic compounds (VOCs) (e.g.
benzene, toluene, and formaldehyde), semivolatile organic compounds (SVOCs) (e.g.
phthalate plasticisers, and pesticides), and human bioeffluents (e.g. carbon dioxide) in the
indoor environment (Wang et al., 2004). This resulted in the emergence of sick building
syndrome (SBS) (Apte et al., 2000; Wang et al., 2004) and elevated pollutant
concentrations had to be reduced by introducing natural or forced ventilation, in order to
reduce indoor air pollution to an acceptable level (Maroni et al., 1995; Graudenz et al.,
2005). Today, mechanical ventilation systems are widely used to maintain a thermally
comfortable environment, as well as acceptable indoor air quality (IAQ) (Yu et al., 2009;
Zhao and Wu, 2009).
Zhao and Wu, (2009) have identified four main factors that affect particle concentration
in the breathing zone: fresh air exchange rate, particle filter efficiency, the type of the
ventilation duct (roughness) and ventilation modes. They also found that enhancing the
filter efficiency or increasing the roughness of ventilaton duct surfaces can reduce indoor
particle pollution.
2.2.1.1.1 Ventilation
Natural ventilation replaces indoor air with fresh outdoor air without using mechanical
power, therefore it can reduce the energy consumed by heating, ventilation and air-
37
conditioning systems in buildings (Jiang et al., 2003). However, the complexities
associated with devising a successful building design that allows adequate natural
ventilation mean that mechanical ventilation is often preferred in many situations.
Ventilation is one of the key factors that affect particle removal and deposition rates
indoors due to air turbulent movement (Jamriska et al., 2000; Howard-Reed et al., 2003;
Wallace et al., 2004a) and many studies have investigated the effect of ventilation on
particle concentrations indoors (Jamriska et al., 2000; Howard-Reed et al., 2003; Wallace
et al., 2004a; Akunne et al., 2006). While Jamriska et al. (2000) investigated the effect of
ventilation on the reduction of submicrometer particle concentration in an occupied office
building, Howard-Reed et al. (2003) and Wallace et al. (2004a) quantified the particle
loss rate in occupied townhouses due to the operation of central fans.
Walsh et al. (1984) also studied the effect of mechanical ventilation on indoor fungal
spore levels, and found that central air conditioning reliably reduces indoor fungal spore
levels by 50% or more, providing that the doors and windows remain closed.
In addition to ventilation, air exchange rate and air movement are also among the main
factors affecting air quality in the indoor environment (Graudenz et al., 2005). Air
exchange rates (AER) are affected by house design and material characteristics, as well as
meteorological conditions and building operational factors (He, 2005 ), and a high AER
has been found to reduce indoor air pollution to an acceptable level (Maroni et al., 1995).
In the subtropical environment of Brisbane, under natural ventilation conditions, air
exchange rates ranged from 2 - 5 h-1, with minimum ventilation between 0.5 - 1.0 h-1
(Morawska and Jamriska, 1994).
38
Fungal Spore Deposition Rates
Spore deposition rates can be estimated in the indoor environment under two contrasting
circumstances – at a low AER (for example, when the ventilation is off) and at a high
AER (for example, when the ventilation is on). When estimating the deposition in a
chamber simulating a typical indoor environment, the spore deposition rates can be
derived using the mass balance equations as follows:
• At a low air exchange rate
70-90% of viable fungi in indoor air fall within the size range 0.54-7.0 µm (Li and Kuo,
1994; DeKoster and Thorne, 1995). For these particle sizes, the deposition rate on indoor
surfaces is determined mainly by gravitational settling velocity. Assuming well-mixed
conditions, the rate of change in indoor concentration for any particle size, with respect to
time, can be expressed using the following mass balance equation (Koutrakis et al., 1992;
Chen et al., 2000; Thatcher et al., 2002; Wallace et al., 2004b; He et al., 2005):
dpidpdpidpodpdpi CCGCf
dtdC
,,,, βαα −−+= 2.1
where α is the air exchange rate (h-1); fdp is the penetration efficiency of the particle
diameter of interest (dimensionless); Co is the outdoor concentration (outside the
chamber) at time t (particles /cm3); G is the generation of particles indoors (particles /cm3
h); Ci is the indoor concentration at time t (particles/cm3); β is the deposition rate
coefficient (h-1); and dp is diameter of the particle of interest (micrometers). From this
equation it can be seen that there is an indirect relationship between indoor and outdoor
particle concentrations, and that the relationship depends on the air exchange rate,
penetration efficiency, deposition rate and indoor particle generation rate. At a low
particle concentration, coagulation can be ignored and chemical reaction and hygroscopic
39
growth can also be ignored, since they were not relevant to the controlled experimental
conditions of this study.
In the absence of the indoor particle sources, Eq. (2.1) can be rewritten as follows:
dpidpdpidpodpdpi CCCf
dtdC
,,,, βαα −−= (2.2)
where the experiment is conducted in a tightly sealed chamber, such that the air exchange
rate (α) is very small and can also be ignored. Alternatively, when the indoor particle
concentrations are significantly higher than the outside level, the contribution from
outdoor sources is very small and can be ignored. Assuming that α and βdp are constants,
the time dependent solution to Eq. (2.2) becomes:
tCC
dpdpi
dpi )()ln()0(,
, βα +−= (2.3)
where the Ci,dp(0) is the peak indoor particle concentration (i.e. t =0). Based on Eq. (2.3),
βdp can be determined by fitting a line to the plot of the natural log of Ci,dp/Ci,dp(0) versus
time and subtracting α from the slope.
• At a high air exchange rate
When using mechanical ventilation, and assuming well-mixed ventilated conditions, the
infiltration from outdoors, other than from ventilation (α), is negligible when compared to
the ventilation rate. When no particle generation or coagulation is occurring in the
chamber during the decay, the rate of change in indoor concentration of any particle size,
with respect to time, can be written as follows:
dpidpdpivdpovdpi CCC
dtdC
,,,, βλλ −−= (2.4)
40
where, λv is ventilation air exchange rate (h-1). If the ventilation system is equipped with a
HEPA filter, the particle infiltration from outdoors can be ignored. Assuming that λv and
βdp are constants, the time dependent solution to Eq. (2.4) becomes:
tCC
dpvdpi
dpi )()ln()0(,
, βλ +−= (2.5)
And again, βdp can be determined by fitting a line to plot of the natural log of Ci,dp/Ci,dp(0)
versus time and subtracting λv from the slope
2.2.2 Fungal Release The variety in composition, dispersal method and size distribution of airborne pollutants
causes them to disperse and be transported differently. For example, polypores rely
mostly upon airborne basidiospores, and millions of spores are produced to increase the
chance of finding a suitable surface on which to be deposited (Kauserud et al., 2008).
Fungi produce conidia or spores that easily become airborne (Larone, 2002), and these
airborne spores are the primary means of fungal propagation (Muilenberg, 1995). Some
spores, however, tend to remain attached to the parent mycelium, and so reducing their
dispersal (Griffin, 1994).
Fungal spore release can be a passive or active process (Burnett, 1976; Muilenberg,
1995). Fungal spores are released passively when the energy is provided by external
sources, such as air currents, rain splash, gravity or insects and other animals (Gregory,
1973; Pasanen et al., 1991; Dix and Webster, 1995; Madelin and Madelin, 1995). Air
currents have been identified as the main cause of passive spore release (Burnett, 1976;
Ingold, 1979).
41
Active fungal spore release occurs when the energy is provided by the fungi (Górny et al.,
2001). This method of release often occurs in ascospores and basidiospores, it requires
moisture or high humidity and it occurs as a result of the osmotic pressure that develops
within the ascus of ascomycetes (Levetin, 1995; Moor-Landecker, 1996). In many fungi,
such as Cladosporium and Penicillium, there is no mechanism of active discharge,
however the spores are freely released into the air (Ingold, 1979).
Fungal spore release is affected by many factors including wind speed, humidity, rain,
building materials, fungal species, ventilation, human activity etc (Muilenberg, 1995).
Spore release also depends on the kind of spore, for example whether it is dry, such as the
spores of Cladosporium, Alternaria, Curvularia, and Paeciliomyces, or wet, like
Fusarium spores. In the dry-spore fungi, release occurs at decreasing relative humidity
and increasing air speed conditions, while wet spores require insects or water splash to be
released. To date, little research has been done on the release of fungal spores in indoor
environments. A study regarding the effect of air flow and humidity found that the
minimum air speed required for spore release is in the range 0.2 - 0.4 m/s, and that the
number of spores increased with increasing wind speed and decreasing relative humidity
(Gregory, 1973). Another study showed that airborne P. chrysogenum concentrations
were significantly higher at 1-1.5 meters (i.e. the adult breathing zone) after walking on
cut pile carpet when compared to loop pile carpet or vinyl tile, confirming the importance
of carpet materials selection (Buttner et al., 2002). Kildesø et al. (2003) investigated the
release of spores from nine different fungal species and found that spore release is
specific to each fungal species, and that the relationship of release (fungal spores) to air
velocity depends on the fungus itself. Górny et al. (2001) revealed that the species of
fungi, texture of the surface, air velocity above the surface and vibration levels all had an
important effect on fungal spores release.
42
2.2.3 Fungal spore transport Once released, spores may enter the air below the boundary layer or above it. If the spores
are released directly above the ground, where the air is still, the spores fall to the ground.
However, if they reach turbulent layers of the atmosphere, they will be dispersed by the
eddies and convection currents in the atmosphere (Burnett, 1976). In a given environment
the length of time a particle remains airborne depends mainly on its size, as well as on its
density, electrical charge, shape and nature of particle surface, ambient air surface and the
availability of impaction surfaces (Muilenberg, 1995). In still air, spores fall to the ground
due to gravity, and for spherical particles, the rate is proportional to the square of the
spore radius (Levetin, 1995). While fungal spore aggregation (as in Penicillium and
Cladosporium) increases effective particle size and consequently increases the rate of fall,
irregular shaped spores have increased surface drag and this delays spore deposition
(Lacey, 1991). Updrafts, which play a significant role in the distance a particle will travel,
have different effects on particle transport depending on the particles’ settling velocity.
While the settling velocity of bacteria is of the order of thousandths of a centimetre per
second, large fungal spores (of around 20 µm) have a settling velocity of centimetres per
second and pollen (of around 100 µm in diameter) settles at tens of centimetres per
second (Muilenberg, 1995). Gregory (Gregory, 1952) suggested that fungal spores of
about 10 μm in length represent a compromise between efficient dispersal and deposition.
Fungal spores are a naturally occurring component of the air turbulent layer and they are
carried by wind both vertically and horizontally. As a result of atmospheric dispersion,
bioaerosols can travel considerable distances (Cox and Wathes, 1995). Fungal spores
have been recovered from altitudes of over 5 km, as they are carried upward thermally,
and they have been carried for thousands of kilometres horizontally (Levetin, 1995).
Many studies have been carried out on the long-distance transport of fungal spores in
43
outdoor environments. One of these studies found that fungal spores were transported
from Yucatan to Geronimo Creek Observatory in smoke from biomass fires, a distance of
1450 km in 2-3 days (Mims and Mims, 2004). Another study showed that several types of
fine fungal spores, which originated in China and Mongolia, were found in the Korean
peninsula, after transport by an Asian dust storm. These spores were found on fine
suspended particle matter (dust) in the size range of 1.1 - 2.1 µm in diameter (Yeo and
Kim, 2002).
2.2.4 Interaction of spores with non-biological aerosols Both outdoor and indoor air contain mixtures of biological aerosols such as fungi and
bacteria, as well as non-biological particles including tobacco smoke, cooking generated
particles, motor vehicle exhaust particles, dust and organic and inorganic gases (CO, SO2,
NO2, O3 etc) (Cox and Wathes, 1995; Baron and Willeke, 2001). When considering the
behaviour, and ultimately the fate of biological particles in the air, it is important to keep
in mind that the concentration of these particles is usually significantly lower than the
concentration of particles that are of non-biological origin (Morawska and Salthammer,
2003b). This implies that there could be various interactions occurring between the two
types of particles, of which the most likely would be coagulation, which results from
particle collisions. This process is strongly dependent on particle concentration and
particle size, and is governed by the rate of diffusion and movement of particles.
The role of interactions between biological and non-biological airborne pollutants in
affecting the dynamics and transport of biological particles is not yet clear. However, it is
considered probable that interactions may occur between biological and non-biological
pollutants, which could affect their behaviour in the air, and ultimately the effect they
have on health. Few studies have investigated whether the non-biological particles act as
carriers for biological particles or vice versa. Fabries et al. (2001) reported that
44
microorganisms are generally attached to non-biological particles in ambient air. A study
in Norway concluded that soot particles in airborne house dust may act as carriers of
several allergens in indoor air (Ormstad et al., 1998). Using an immuno-gold labelling
technique, they found Fel d1 (cat) allergen heavily attached to the soot particles of indoor
suspended particulate matter samples. The cytoplasmic contents of spores and pollen are
also often found adhered to motor vehicle emission material and crustal matter.
Therefore, these may act as carriers for dispersed cytoplasmic allergenic material released
from pollen and fungal spores (Glikson et al., 1995), and they may also serve as a
substrate for numerous species of viable spores, especially the soil fungus Aspergillus.
Shinn et al. (2000) also suggested that Aspergillus sydowii spores were carried by dust
blown from the Saharan deserts across the Atlantic Ocean, where they reached the
Caribbean islands and caused aspergillosis disease in the entire Caribbean sea fan.
2.2.5 Deposition Airborne particles are removed from indoor environments by mechanical and passive
ventilation, deposition and sedimentation onto horizontal surfaces (Schneider, 1991). One
of the most important elements of particle dynamics is the deposition of aerosol particles
in the indoor air, onto surfaces (Lai, 2002; Zhao and Wu, 2007). For example, the rates of
airborne particle removal by surface deposition can be as large as or larger than removal
through typical ventilation rates and therefore, keeping surfaces clean is just as important
in controlling particle contamination in indoor air as maintaining adequate ventilation
(Schneider, 1991). In addition, the deposition of airborne particulate matter can lead to
the soiling and damage of indoor surfaces, including, for example, works of art kept in the
home or museums (Roshanaei and Braaten, 1996; Spolnik et al., 2007).
The factors that affect particle deposition include convection, Brownian diffusion,
turbulence, sedimentation, inertial effect, thermophoresis, electrophoresis and surface
45
geometry (Chen and Chan, 2008). Other factors can also influence particle deposition in
indoor environments, including air flow conditions near the wall surfaces, surface
roughness and particle concentration distribution (Lai et al., 2002; Zhao and Wu, 2007).
A study by Zhao and Wu (2007) found that rougher surfaces and a larger friction velocity
near the wall surfaces may lead to a larger particle deposition velocity, but only when the
particle size is small. They also found that particle concentration distribution, which may
vary greatly even for the same particle source and air exchange rate, can result in
variation in the particle deposition flux. Lai et al. (2002) found that the ratio of particle
deposition on rough surfaces, relative to that on smooth surfaces, increased with particle
size and magnitude of airflow. In another study, Lange et al. (1994) found that deposition
velocities increased with particle size and the amount of furniture, and that deposition
onto walls and ceilings increased with increasing size as well.
Many studies have been conducted to establish models for use in predicting particle
deposition velocities indoors. These studies can be categorised into two groups -
theoretical studies (Nazaroff et al., 1990; Kulmala et al., 1999; Schneider et al., 1999) and
studies of particle deposition using numerical methods (Lu and Howarth, 1996; Zhao et
al., 2004a; Zhao et al., 2004b). In general, the deposition rate, as derived by model
estimates, could be significantly different from those found in experimental studies,
particularly for particles smaller than about 0.5 µm (Morawska and Salthammer, 2003a).
Only one of the abovementioned numerical studies used experimental data from the
literature to validate their numerical model (Zhao et al., 2004a).
A large number of studies has been conducted to determine the deposition rates of non-
biological aerosols in indoor environments (Byrne et al., 1995; Thatcher and Layton,
1995; Fogh et al., 1997; Long et al., 2001; Mosley et al., 2001; Vette et al., 2001;
46
Thatcher et al., 2002; Howard-Reed et al., 2003; Ferro et al., 2004; He et al., 2004;
Wallace et al., 2004a; He et al., 2005). However, very little work has been done in
relation to the deposition rate of fungal spores (Reck et al., 2002). A study conducted by
Reck et al. (2002) investigated experimentally and numerically, the deposition of spoilage
fungi on a petri dish, on a surface and on a warm box-shaped object placed in a food-
processing environment. In another study (Sawyer et al., 1994), the settling velocity of
Zoophtora radicans, Conidiobolus obscurus, and Conidiobolus thromboides fungal
spores were measured by allowing fungal cultures to sporulate into a glass chamber. The
falling conidia were then observed using video microscopy and the images were analysed
using a computerised motion analysis system, in order to determine the settling velocities
(Sawyer et al., 1994).
2.2.6 Summary It is expected that the dynamics of particles in the air, including bioaerosols, will be
governed to a large extent by the physical characteristics of the particles, of which size is
one of the most important. Some of the physico-chemical processes of bioaerosol
dynamics in indoor environments include fungal release, transport, interaction of spores
with non-biological aerosols and fungal spore deposition. The fate of the smallest
airborne particles is governed by Brownian motion, while larger particles are subject to
gravitational and inertial forces. In most studies, the deposition rate is derived by using
the mass balance equation.
Ventilation is one of the key factors that affect particle deposition rates indoors due to
turbulent air movement. Many studies found that elevated contaminant concentrations are
reduced in buildings by the use of natural or forced ventilation, and that high ventilation
rates reduce indoor air pollution to a low level.
47
Fungal spore release may be a passive or an active process. It is affected by many factors,
such as wind speed, humidity, rain, building materials, fungal species, ventilation,
vibration and human activity. One study on the effect of air flow and humidity found that
the minimum air speed required for spore release is in the range 0.2 - 0.4 m/s, and that the
number of spores increased with increasing air speed.
Many studies have been conducted on the long-distance transport of fungal spores.
Fungal spores are commonly found in the turbulent air layer and they are carried by wind
both vertically and horizontally. While fungal spores have been recovered from altitudes
greater than 5 km, they have also been found to be carried for thousands of kilometres
horizontally. In contrast to long-distance transport, very little work has been done on the
transport of fungal spores in indoor environments to date.
As the concentration of biological airborne particles, including fungi, is significantly
lower than the concentration of particles that are of a non-biological origin, various
interactions can occur between the two types of particles, of which the most likely is
coagulation. This interaction may not only affect the particles’ behaviour in the air, but it
may ultimately affect the impact they have on health. Few studies have actually
investigated whether non-biological particles act as carriers for biological particles, or
vice versa.
Aerosol particles in indoor air often deposit onto surfaces and deposition is one of the
most important elements of the dynamics of airborne particles. For example, the removal
of airborne particles from the air by surface deposition can be as large as or larger than
that caused by typical ventilation rates. The factors that affect particle deposition rates
include convection, Brownian diffusion, turbulence, sedimentation, inertial effects,
thermophoresis, electrophoresis and surface geometry, as well as the air flow conditions
48
near the wall surfaces, surface roughness and particle concentration distribution. While
numerous published papers have quantified the deposition rates of non-biological
particles, very little work has been done on the deposition rate of fungal spores and no
studies have been conducted to estimate the deposition rates of fungal spores in a room-
sized chamber. Similarly, no published studies have examined the effect of ventilation on
fungal propagule deposition rates.
2.3 Aerosol and bioaerosol sampling techniques and samplers
2.3.1 Introduction Two important characteristics distinguish current development in aerosol measurement
technology from that which occurred before 1960s; firstly, new developments exploited
the experience and the knowledge obtained in previous years and secondly, developments
occurred as a result of the newly obtained knowledge and techniques in the fields of
microelectronics, computer, laser, analytical chemistry and analytical electron
microscopy (after the 1960s) (Willeke and Baron, 1993; Spurny, 1999a). These
developments contributed to improvements in the technology used for cascade impactors,
electrical aerosol mobility analysers, and optical particle counters and analysers, and they
also contributed to the development of new methods and techniques, such as analytical
aerosol filters, chromatography, mass spectrometry, and plasma and laser spectroscopy
(Spurny, 1999a).
Sampling is the most critical factor when conducting aerosol measurements in indoor
environments and choosing the most appropriate sampler is not always an easy task
(Berlinger et al., 2007). During the last two decades, bioaerosol sampling technology has
improved significantly, however to date, there are no standard bioaerosol sampling
methods (Grinshpun and Clark, 2005). Many studies have reported that certain
49
environmental conditions are required in order to accurately assess fungal exposure. For
example, Ren et al. (2001) reported that the presence of fungal propagules in indoor air
cannot be reliably predicted using housing characteristics as described by questionnaires.
Instead, actual measurements are required. Verhoeff et al. (2007) found that the results of
quantitatively and qualitatively measured mold propagules in house dust depend greatly
on the analytic methods used. They also found that a single measurement of fungal
propagules in settled house dust does not provide a reliable indication of the potential
exposure to fungi in indoor environments (Verhoeff et al., 2007).
2.3.2 Sampling methods To detect, quantify and identify bioaerosol release from sources, as well as to assay
human exposure to biological agents and monitor the effectiveness of control strategies,
biological aerosol samples need to be collected. Sample collection may include visual
observation, air sampling, bulk sampling or surface sampling (Burge, 1995; Martyny et
al., 1999; Willeke and Macher, 1999b). Observation sampling consist of walking through
the environment/s and using human senses to assess the biological status of the area
(Burge, 1995). While bulk samples consist of portions of environmental materials, such
as settled dust, a piece of wallboard, a carpet segment or a piece of duct lining, surface
samples are collected by placing a suction device, contact plate or adhesive tape onto
surfaces and/or washing the area of interest with a wetted swab or cheesecloth (Martyny
et al., 1999). In contrast, air sampling is the most widely utilised technique for conducting
aerosol and bioaerosol measurements (Baron and Willeke, 2001).
2.3.3 Bioaerosal air samples: air samplers and techniques
Two types of methods are used for sampling of bioaerosols, conventional methods and
real-time methods.
50
2.3.3.1 Conventional methods of bioaerosol air sampling
Conventional methods such as impaction, impingement and filtration are time consuming
and labour intensive. They generally consist of two stages: the field sample collection,
followed by off-line laboratory analysis (Agranovski, 2004). Conventional methods
provide average data for indoor fungal particle concentrations over a period of time such
as 2 hours, 24 hours or 48 hours, and therefore no information can be obtained about
time-related particle concentrations. For example, grab samples of indoor fungal
concentration levels do not effectively represent actual concentrations (Kamens et al.,
1991; Lyons and Morawska, 1996). Data related to short time-increments (20 second or
less) will be very useful in identifying specific household activities that may generate
high levels of bioaerosols. In addition, short-term peaks (burst phenomenon) which can
have a significant impact on human health, may not be detected (Michaels and Kleinman,
2000; He, 2005 ; Falvey and Streifel, 2007).
Particles of biological origin are subjected to the same principles as other aerosols when it
comes to achieving good sample collections, however their biological properties also
mean that they require certain other aspects of collection to be taken into consideration.
For example, overall biological sampler efficiency also depends on inlet sampling
efficiency, particle removal efficiency and biological recovery efficiency (Willeke and
Macher, 1999b). The efficiency of conventional bioaerosol samplers is measured by how
well the biological properties of the particles, such as culturability and morphological
details, are retained (Baron and Willeke, 2001). The survival of bioaerosol particles
depends on impaction velocity (higher velocities, resulting more stress), sampling time (a
long sample time increase desiccation) and the particle’s internal characteristics (pollen
grain and microbial spores are more protected against stress than vegetative cells) (Cox
and Wathes, 1995; Baron and Willeke, 2001).
51
Different physical forces can be used to separate and collect particles from the air stream,
using a wide range of samples. Samplers may be used for the collection of bioaerosols
from both indoor and outdoor environments. Each sampler has specific advantages and
disadvantages, and the choice of sampler depends on the biological agent of interest, the
study objectives and the environmental conditions (Agranovski, 2004). Hence, choosing
the proper instrument for a particular application is very important. Currently, bioaerosols
are commonly collected using active sampling techniques, such as impaction,
impingement or deposition on filters (Han and Mainelis, 2008), as described in the
following sections.
2.3.3.1.1 Inertial impaction
Inertial impaction is the most widely used bioaerosol particle collection method. The
impaction method is used to separate particles (fungal spores in this case) from the air and
collect them on a selected media (i.e. the agar impaction surface). The airborne particles
sucked through the air jet, are focussed onto the agar (collection surface) and the larger
particles which cannot follow the air jet impact the agar. The principle of inertial
classification is that during a rapid change in the direction of airstream, a particle with
sufficient inertia can cross the airstream and escape from the flow, while those particles
with less inertia remain in the flow. Particle inertia depends on the size, density and
velocity of the particle, as well as on the physical properties of the impactor. Impactor
performance is often described by the cut-off size (d50), which denotes the particle size at
which 50% of those particles are collected by the impactor onto the collection surface
growth-medium. To ensure the collection of the majority of microorganism particles, an
impactor with a d50 smaller than the size of interest should be selected (Yao and Mainelis,
2006). Examples of samplers that apply this principle are single and multiple stage
impactors (1-, 2-, 6 stage, the most common of which is the Andersen six-stage impacting
52
sampler), the Air-O-Cell, Burkard samplers, the slit-to-agar biological sampler, the Reuter
centrifugal air sampler (RCS), the Surface Air System (SAS) and cyclone samplers
(Griffiths and Stewart, 1999; Macher, 1999; Willeke and Macher, 1999b; Grinshpun et
al., 2005; Ho et al., 2005).
The main advantage of these methods is the discrimination between particle sizes. On the
other hand, disadvantages lie in the desiccation of collected microorganisms over
prolonged sampling time, underestimating the number of airborne particles due to particle
bounce or the deposition of more than one particle on the same site, and the fact that the
effective concentration range of the majority of agar plate impactors is below 104 colony
forming units per cubic meter (CFU/m3) (Cox and Wathes, 1995).
2.3.3.1.2 Liquid impingement
Particle collection in the liquid impingement method is achieved primarily by inertial
impaction into liquid, which is assisted by particle diffusion within the bubbles (Lin et al.,
1997; Willeke and Macher, 1999b; Baron and Willeke, 2001). All-glass impingers (AGI)
were designed specially for microorganism collection and the commercial names of these
samplers are AGI-4, AGI-30, three stage impinger SKC bio-sampler and a tangential
impactor (Willeke and Macher, 1999b). Of these, the AGI-30 was reported to be suitable
for fungal assays (Cox and Wathes, 1995). The advantages of using this method when
collecting biological aerosols are its ability to be used over a wide range of concentrations
and its ability to help prevent the dehydration of biological agents, as well as the fact that
it is inexpensive, simple to use and it is compatible with a wide range of analytical
(microbiological) techniques used to analyse the samples (Agranovski, 2004). On the
other hand, the disadvantages of this method, which result in reduction of the collection
efficiency of these impingers, include the evaporation of the liquid during impingement, a
53
high air flow rate which leads to particles bouncing off the bottom, and the bursting of the
bubbles which may cause particle re-aerosolization (Grinshpun et al., 1996).
The SKC bio-sampler has been developed to prolong the sampling period, in comparison
to other conventional samplers (i.e., AGI-30) (Willeke et al., 1998). The conventional
sampling of bioaerosols into impingers is often performed with a low viscosity liquid
(which evaporate quickly), and therefore, not only the sampling time is limited 15-30
min, but the bioefficiency also decreases rapidly with time. Incontrast, the liquid used in
the SKC bio-sampler lasts for a longer period of time and the collection efficiency only
decreases moderately with time. under these conditions, it is much more likely the bio-
sampler will be able to detect culturable bioaerosols at lower ambient concentrations (Lin
et al., 1999). In the bio-sampler, the airborne bioaerosols are drawn into three nozzles,
projected at an angle and then collected into the liquid by a combination impaction and
centrifugal forces (Lin et al., 1999).
Although the impinger method is not the most efficient for use on hydrophobic bacteria or
fungal spores, as opposed to impactors (Willeke and Macher, 1999b), the microbal stress
is less when operating at the same sampling velocity (Grinshpun et al., 1996),
2.3.3.1.3 Filtration
As a result of its simplicity and economy, filtration is a widely utilised technique for
aerosol sampling. During the filtration process, the particles are separated from the air by
directing the air stream through a porous medium (i.e. the filter). The filter contains
numerous uniform sized pores, ranging from 0.2-1 µm in diameter, and the collection of
aerosols and/or bioaerosols on the filter depends on pore size, as well as particle size and
airflow rate (Macher, 1999; Lee and Mukund, 2001). Filtration is an efficient method for
54
collecting a number of biological agents, such as endotoxins, antigens, ergosterol and
other biological components (Willeke and Macher, 1999b).
There are many types of filters, such as fibrous filters (cellulose, glass, quartz, asbestos
and plastic fibrous filters), membrane filters (polyvinyl chloride, nylon, Nuclepore filter
and polycarbonate filter) and granular bed filters, of which the fibrous filter is the most
commonly used (Lippmann, 1989). Polycarbonate and cellulose ester filters are also
widely used for bioaerosol collection, as are cassette filters (Willeke and Macher, 1999b).
Particle collection on the filter is achieved by sifting out those particles larger than the
filters pore size, through interception or inertial impaction (Macher, 1999). Diffusional
deposition, electrical attraction and gravitational attraction are other physical methods
used for removing particles from a gas stream using filters. The predominant mechanism
of the filtration method used depends on flow rate and the nature of both the filter and the
aerosol (Lippmann, 1989).
There are many advantages for using filtration techniques, in that the instruments are
often portable, inexpensive and can be applied over wide range of sizes. On the other
hand, the disadvantages include the desiccation of the sample in hot or dry weather and
the decrease in spore culturability with increased sampling time (Wang et al., 2001b;
Agranovski, 2004).
2.3.3.1.4 Electrostatic precipitator
In electrostatic precipitators, airborne particles are charged electrically and then removed
from the air stream by an electrostatic field (Cox and Wathes, 1995; Han and Mainelis,
2008). This method was found to be suitable for the collection of the airborne
microorganisms (Grinshpun et al., 1996). Examples of samplers using this technique are
55
the Litton-type large volume liquid electrostatic aerosol precipitator sampler (LVAP) and
the liquid electrostatic aerosol precipitator sampler (LEAP) (Cox and Wathes, 1995).
The advantages of this method are the collection of biological agents in high volumes
(1000 L/min), as well as the ability to collect samples into liquid or onto agar in plastic
strips (Agranovski, 2004). The electrostatic precipitation technique is also potentially less
damaging to the microorganisms than the bioaerosol impactors and impingers operating
under comparable sampling conditions (Mainelis et al., 2002). However, the
disadvantages of this method are its complexity: the expense and difficulty associated
with sterilisation and that survival of microorganisms may be adversely affected by an
increased sampling time (Grinshpun et al., 1996; Agranovski, 2004).
2.3.3.1.5 Gravitational sedimentation
Open Petri dishes can also be used as samplers for the collection of bioaerosols. This
method is simple, inexpensive and does not need a power source, although it is only
useful for providing qualitative information and the results are significantly affected by
particle size and air movement (Agranovski, 2004).
Common conventional bioaerosol samplers
The Andersen six-stage impacting sampler and the all glass AGI-30 impinger are the two
most commonly used devices for microbial air sampling (Maier et al., 2000).
• Andersen Impactor
The Andersen impactor, a type of cascade impactor, is the most commonly used impactor
for bioaerosol sampling and it contains several impactors (normally six or eight) with
different cut-off sizes. The upper stage has the largest cut-off size, which decreases
gradually until reaching its minimum in the lower stage. The cut-off size decreases in this
manner as a result of decreasing orifice diameters (nozzles sizes) in each stage. Each
56
stage collects particles larger than its cut-off size and the last stage is often followed by a
filter to capture all remaining particles smaller than the last stage cut-off size. The
Andersen ambient cascade impactor has eight stages, with 400 holes in each stage, and it
works at a flow rate of 28.3 L/min, with a d50 (cut-off size) ranging from 0.4-9 µm
(Hinds, 1982; Jensen and Schafer, 1998). As such, it is suitable for investigating fungal
spores with a size equal or less than 9 µm.
• AGI-30 impinger
The conventional AGI-30 is recommended by the American Conference of Industrial
Hygienists (ACGIH) as a standard or reference device for collecting viable bioaerosols
onto a liquid media. Most impingers are designed to draw air through a curved tube to
imitate the nasal pathway. The air then goes through a nozzle to impact into the medium.
The AGI-30 can capture particles from 0.3 – 15 µm, at a flow rate of 12.5 L/min. Liquid
media such as deionised sterilised water or 0.3 mM phosphate-buffered dilution water are
used for collection. Protein and antifoam may be added to the collection medium to help
minimise loss of particles, as well as prevent bacterial injury (Jensen and Schafer, 1998).
After sampling, a defined amount of the collection fluid (i.e. 10, 20, 50, or 100 µl) is
spread on a suitable medium and incubated for identification and enumeration.
2.3.3.2 Real-time methods in bioaerosol air sampling
Recently, several studies have reported on the use of various instruments for the
measurement of fluorescence spectra as a technique for the real-time detection of single
viable airborne bioaerosols. These studies can be divided into three categories. The first
category includes studies that aimed to investigate instruments capable of differentiating
between biological and non-biological aerosols, such as Fluorescence Spectrum Analyser
and the Ultraviolet Aerodynamic Particle Sizer (UVAPS) (Hill et al., 1995; Pinnick et al.,
57
1995; Chen et al., 1996; Nachman et al., 1996; Hariston et al., 1997; Pinnick et al., 1998;
Ho et al., 1999; Brosseau et al., 2000; Kaye et al., 2000; Pan et al., 2003). The second
category of study includes studies that investigated instruments with the ability to
characterise particle composition in order to discriminate between different bioaerosols
themselves (Cheng et al., 1999; Pan et al., 1999; Seaver et al., 1999; Weichert et al.,
2002; Sivaprakasam et al., 2004). Some of these studies utilised multiple UV excitation
wavelengths to produce more than one fluorescence spectrum for each species under
investigation (Cheng et al., 1999; Sivaprakasam et al., 2004). The third group of studies
combined the UVAPS with other technologies, such as wet chemistry technology so that
after the aerosols of interest were detected by UVAPS, samples were collected for further
analysis, in order to identify the microorganisms using the wet chemistry technique (Ho,
2002).
Despite these investigations, the measurement and characterisation of bioaerosols is still a
very challenging problem (Grinshpun and Clark, 2005). To date, the UVAPS has been the
main instrument used for the real-time detection of viable bioaerosols and its operation is
based on the excitation and emission of the auto-fluorescent biomolecules which exist in
most bioaerosols, as outlined below:
2.3.3.2.1 Fluorescence and biochemical fluorophores
The term “fluorescence” was coined in 1852 by G.G. Stokes, when the mineral fluorspar
(CaF2) was observed to emit visible light after being illuminated by UV light (Cooper,
2004). When the molecules in the fluorescent compound absorb ultraviolet and visible
light (photons), they are exited from ground state, to reach an excited state. This excited
energy can be released by one or more of the following mechanisms: (1) it can decay non-
radiatively to the ground state and the energy of the excited electron is dissipated as heat;
(2) the molecules return to ground state with the emission of light or fluorescence; (3) the
58
excited energy may be transferred by resonance energy transfer to a neighbouring
molecule; or (4) this energy may change the reduction potential of the absorbing
molecules and turn it into an electron donor (Perry and Staley, 1997; Cooper, 2004).
Fluorescence radiation is characterised by the quantum yield (Cooper, 2004). Usually the
absorbed photon is in the ultraviolet range, while the emitted light is in the visible range.
While the fluorophore excitation at different wavelengths does not change the emission
profile, it does produce variations in fluorescence emission intensity. The intensity of the
emitted fluorescence light is at its maximum when the used excitation light is at
maximum absorption wavelength (Agranovski, 2004).
Depending on the type of fluorescence phenomena, fluorescence microscopy may be used
to examine materials that fluoresce, either directly, if the fluorescence is natural, or by
viewing the fluorescence resulting from an attached fluorescent compound such as an
antibody that binds the object of interest, or by viewing the fluorescence that results when
fluorescent dyes, such as acridine orange, are used for staining microorganisms (Perry
and Staley, 1997; Nester et al., 2004). These fluorescence microscopy techniques have
many important applications in the medical microbiology field (Nester et al., 2004).
2.3.3.2.2 Organism viability and the natural auto-fluorescence
The term “viable” means alive and able to grow, while the term “culturable” is used for
viable organisms that can be recovered using an artificial culture medium (Muilenberg,
1995). In particular, microbes may be viable but non-culturable (Maier et al., 2000).
Viability is highly dependent on the type of microorganism, the environment and the
period of time the organism spends in the environment. The most important
environmental factors that have been shown to influence the ability of a microorganism to
survive are relative humidity and temperature. Oxygen content, specific ions, UV
59
radiation and various pollutants are also known to result in the loss of biological activity
(Maier et al., 2000).
The viable cells of most organisms, including fungi, have natural auto-fluorescence due to
biochemical fluorophores, such as the reduced fluorescent co-enzymes nicotinamide-
adenine dinucleotide (NADH) and nicotinamide-adenine dinucleotide phosphate
(NADPH), the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide
(FAD), and metabolic function riboflavin (vitamin B2) (Li et al., 1991; Brosseau et al.,
2000; Billinton and Knight, 2001). Riboflavin is of central importance in the respiratory
chain. Living organisms gain most of their energy from oxidation-reduction reactions and
these processes involve the transfer of electrons via an electron transport chain made up
of proton- and electron-carrying substances, such as riboflavin. Riboflavin also acts as a
co-enzyme for respiratory enzymes and it is also used in the metabolism of amino acids,
carbohydrates and fatty acids (Agranovski, 2004). In fungi, the catabolism of D-xylose
and L-arabinose pentose sugars to D-xylulose-5-phosphate go through oxidation and
reduction reactions in which the reductions are mainly NADPH- linked and the oxidations
are NAD+ linked (Panagiotou et al., 2005). NAD(P)H also plays a very important role in
the formation of the energy-rich adenosine.
The fluorophore biological molecules, found within microorganisms that contain natural
fluorophores, emit photons after being excited in the ultraviolet region (Cantor et al.,
1980). The maximum excitation and emission peaks for the co-enzymes NAD(P)H are
360 nm and 460 nm, respectively (Dellinger et al., 1998; Billinton and Knight, 2001).
Flavins’ maximum excitation peaks are 360 nm and 445 - 470 nm (Dellinger et al., 1998)
or 385 nm (Li et al., 1991), with a maximum fluorescence peak at 530 nm (Billinton and
60
Knight, 2001) or 525 nm as in Li et al. (1991). NAD(P)H and flavins are largely
responsible for the fungal fluorescence detected by the UVAPS (Agranovski, 2004).
2.3.3.2.3 Ultraviolet Aerodynamic Particle Sizer (UVAPS)
The Ultraviolet Aerodynamic Particle Sizer (UVAPS) (Model 3312, TSI, St Paul, MN) is
a real-time instrument designed for monitoring bioaerosols (Hariston et al., 1997; TSI-
Incorporated, 2000) (Figure 2.1). Besides the real-time fluorescence of airborne particles
within a size range 0.5-15 µm, the UVAPS can provide a number size distribution for
particles with aerodynamic diameters of 0.5-15 µm, as well as scattered light intensities
for these particles.
The UVAPS is constructed by incorporating an Ultraviolet (UV) laser into an
Aerodynamic Particle Sizer (APS). The sample flow path is illustrated in Figure 2.2 and
the overall operation of UVAPS is as follows: after the aerosol is drawn into the inlet, it is
split into a sample, which flows through the inner zone (sample flow), while the
remaining air passes through the outer zone (sheath flow). Once the sample flow passes
through the orifice, it is reunited with the sheath flow and once in the jet, the smaller
particles accelerate faster than the larger ones. Aerodynamic sizing is performed by
measuring the time-of-flight of individual particles when they pass through two laser
beams produced by a 680 nm red diode laser. The scattered light pulses produced by the
particles are then converted, by an avalanche photodiode (APD) and an ellipsoidal mirror,
into electrical pulses. Based on the calibration table, the UVAPS then converts each time-
of-flight measurement into an aerodynamic particle diameter and the obtained particles
for each size are classified into different channels. The UV beam (produced by ultraviolet
laser aimed slightly below the two red laser beams) then illuminates the particles at the
correct time and a certain fluorescence wavelength emitted from the particle is detected
by UVAPS. The resulting signal is converted by the photomultiplier tube (PMT) to
61
electrical signals. The fluorescence is produced by exciting particles with an UV laser
beam at an excitation wavelength of 355 nm and emission light detection at 420 and 575
nm.
Under these conditions, the measured fluorescence is considered specific to living
microorganisms (Hariston et al., 1997; TSI-Incorporated, 2000; Agranovski et al., 2003a).
However, the fluorescence of the excited biomolecules has been found to be strongly
affected by environmental and biomolecule-related factors. For example, Agranovski et
al. (2003b) found that bacterial stress has a large impact on the fluorescence properties of
biomolecules. Huber et al. (2000) also demonstrated that the fluorescence spectra for free
NADH and that bound to proteins were in fact different.
The main goal of the UVAPS is to distinguish particles of biological origin from the vast
array of environmental particles that exist in the air and quantify them. Unfortunately,
like many other real-time samplers, the UVAPS cannot classify different genus or
species, nor can it separate the microorganisms for further analysis (Górny, 2004). The
UVAPS is also not selective for specific microbial fluorescent molecules, since non-
microbial blanks such as agar washings, peptone water and broth media produce
relatively strong fluorescent signals (Agranovski and Ristovski, 2005).
62
Figure 2.1: The Ultraviolet Aerodynamic Particle Sizer (UVAPS) (Model 3312, TSI, St. Paul, MN)
Figure 2.2: Aerosol flow through the UVAPS.
Total Flow
Outer Nozzle Sheath Flow(4 L/min)
AcceleratingOrifice Nozzle
Transducers
Total-Flow Pump
Filter
Inner Nozzle/Sample Flow(1 L/min)
Aerosol In
AbsolutePressure
Transducer
Beam-Shaping Optics (5 L/min)
EllipticalMirror
BeamDump
UV Reflector
UV Beam Input
Pressure
Pump
Sheath-Flow
Pressure Total-Flow
Sheath-Flow
Collimated Diode
Orifice .
Transducer
63
2.3.4 Summary Particles of biological origin are subject to the same principles as other aerosols when it
comes to achieving good sample collection, however their biological properties also mean
that they require certain other aspects of collection to be taken into consideration. For
example, overall biological sampler efficiency also depends on inlet sampling efficiency,
particle removal efficiency and biological recovery efficiency.
Conventional methods such as impaction, impingement and filtration are both time
consuming and labour intensive. They generally consist of two stages: the field sample
collection, followed by off-line laboratory analysis. Conventional methods only provide
average data for indoor fungal particle concentrations, over a short period (hours), and so
it is difficult to estimate the risk of fungal exposure using such type of data. The most
common conventional samplers used in bioaerosol sampling are the Andersen impactor
and the AGI-30.
The Ultraviolet Aerodynamic Particle Sizer (UVAPS) is a real-time instrument designed
for monitoring bioaerosols. It provides real-time fluorescence signals and accurate count
size distribution data for airborne particles with aerodynamic diameters between 0.5-15
µm, as well as scattered light intensities for these particles. Viable cells of most
organisms, including fungi, have a natural auto-fluorescence due to biochemical
fluorophores such as NADH, NADPH and metabolic riboflavin. When fluorophore
molecules absorb ultraviolet and visible light (photons), they are exited from the ground
state to an excited state and the energy of the excited electron emits fluorescence, which
is measured by instruments such as the UVAPS. In order to develop a full understanding
of the capabilities and limitations of the UVAPS when investigating airborne fungal
spores, further research, characterisation and validation of the instrument is required. In
addition, while many studies have investigated the ability of the UVAPS to measure
64
viable airborne bacteria, none have investigated its ability to monitor and measure fungal
propagules.
2.4 Knowledge Gaps
1. To date, little work has been done to investigate the interactions and associations
between particles of biological and non-biological origins. Instead, the physical,
chemical and biological properties of particles have mostly been studied in isolation.
Few studies have investigated whether non-biological particles act as carriers for
biological particles, or vice versa and the role of interactions between biological and
non-biological airborne pollutants in affecting the dynamics and transportation of
biological particles is not yet clear.
2. Despite the role biological agents play in atmospheric systems, there has been very
little research done on their dynamic behaviour in indoor environments. Few studies
have investigated the characteristics of fungal spore release and even fewer have
investigated the transport of fungal spores in indoor environments. The very limited
studies on fungal spore transport are still far from establishing a model for their
transport in indoor air.
3. The deposition of particles on indoor surfaces is one of the most important elements of
particle dynamics. While numerous published papers have estimated the deposition
rates of coarse, fine and ultrafine non-biological particles, very little work has been
done with regard to the deposition rate of fungal spores, nor have the deposition rates
of fungal spores in a room-sized chamber been investigated. In the same manner,
several studies have investigated the effect of ventilation on particle deposition
65
indoors, however none of them have examined the effect of ventilation on the particle
deposition rates for bioaerosols.
4. While previous studies on fungal fragmentation have investigated the effect of air
velocity on fungal fragment release, they did not include a wide range of air velocities.
In addition, they did not investigate the effect of different fungal releasing methods on
the characteristics and dynamics of fungal fragment release.
5. The actual mechanisms involved in the fungal fragmentation process remain unclear
and in order to control this process, further investigations need to be conducted. On the
other hand, the full scale of fungal spore size distribution after fragmentation is also
yet to be studied.
6. While many studies investigate the ability of the UVAPS instrument to measure viable
airborne bacteria, none have investigated its ability to measure fungal spores or its
ability to distinguish between fungal species in the laboratory or in the field. There is
still a need for further characterisation and validation of the instrument in terms of its
selectivity, counting efficiency and sensitivity towards fungi, and more research is
necessary to develop a full understanding of the capabilities and limitations of its
application for studies of airborne fungal spores.
7. In spite of the hundreds of different samplers used for testing aerosolised micro-
organisms and/or non–viable particles, there is still a need for standardised methods
and recommended sampling protocols, in order to increase the accuracy and
reproducibility when sampling microbiological aerosols. It is also important to develop
standardised protocols for performing indoor assessments of airborne fungi, in order to
overcome the difficulty of comparing the results from different studies.
66
8. There is insufficient information available about many of the biologically derived
airborne contaminants to determine the concentration thresholds under which the
substance does not cause adverse health effects. A broad database of fungal spore
levels needs to be constructed for a variety of indoor environments and seasonal
conditions, in order to help further protect people from the serious health hazards
related to fungal spores.
9. The current information available on fungal spores indoors, both in terms of mass
concentration and number concentration, is very limited and there is also lack of
information on the spatial and temporal, or short-term, variations of fungal particle
concentrations indoors. Thus, there is a significant need to conduct time-series
monitoring of indoor fungal particles, in order to provide information on the impact of
temporal fluctuations in particle concentration from indoor particle sources. This data
would not only help to improve scientific understanding, but it would also serve to
improve future assessments of human exposure to airborne fungal particles in indoor
environments.
67
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CHAPTER 3 : Performance of UVAPS with Respect to detection of airborne fungi
Hussein Kanaania, Megan Hargreavesa,b, , Jim Smithb, Zoran Ristovskia Victoria
Agranovskia, and Lidia Morawskaa
aInternational Laboratory for Air Quality and Health, Queensland University of
Technology, Brisbane, QLD, Australia
bSchool of Life Sciences, Queensland University of Technology, Brisbane, QLD,
Australia
(2008) Journal of Aerosol Science 39: 175-189
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Statement of Joint Authorship
Title: Performance of UVAPS with respect to detection of airborne fungi
Authors: Hussein Kanaani, Megan Hargreaves, Jim Smith, Zoran Ristovski, Victoria
Agranovski and Lidia Morawska
Hussein Kanaani: Developed experimental design; conducted experiments; analysed and
interpreted data and wrote the paper.
Megan Hargreaves: Contributed to experimental design; interpreted data and offered
editorial comments during paper writing.
Jim Smith: Contributed to experimental design and offered editorial comments during
paper writing.
Zoran Ristovski: Contributed to experimental design and interpreted data; provided
technical assistance and helped in solving problems during the experiment.
Victoria Agranovski: Offered advice and editorial comments throughout preparation of
the final version of the manuscript.
Lidia Morawska: Contributed to experimental design, helped in solving problems during
the experiment; interpreted data and offered editorial comments during paper writing.
93
Abstract
This paper studies the detection limit, selectivity and counting efficiency of an Ultraviolet
Aerodynamic Particle Sizer Spectrometer (UVAPS) with regard to aerosolized fungal
spores. The study demonstrated the ability of the instrument for detection and
measurement of fungal spores under controlled conditions. A reasonable correlation was
found between the UVAPS and the AGI-30 impinger in measuring the aerosol fungal
spore concentrations under investigation: Penicillium and Aspergillus niger (r = 0.911, p
< 0.005 and r = 0.882, p < 0.05, respectively). A linear relationship between total particle
concentration and fluorescent particle concentration was found in the range from 0 to 70
particles/cm3. Its lower detection limit was found to be 0.01 particles/cm3. The dry
generation method which was used for generating fungal spores has proved to be
reproducible and easy to control, as well as simple and inexpensive.
Keywords: fungal spores; real-time monitoring; counting efficiency; spore release; air flow rate
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3.1 Introduction
There is increasing scientific and medical evidence that exposure to biological aerosols
has significant health implications (Dales et al., 1991; Koskinen et al., 1997; Verhoeff
and Burge, 1997a). Diseases like tuberculosis, diphtheria and legionellosis are caused by
infectious microorganisms that are transmitted via airborne routes and deposited in the
respiratory tract (Maus et al., 2001). Microbial spores and other microbial components
can cause hypersensitivity in susceptible people (Nester et al., 2004). Fungi are
responsible for many allergic conditions in humans such as asthma, rhinitis, allergic
bronchopulmonary mycoses and hypersensitivity pneumonitis (Vijay et al., 1999b).
Infection is another way for fungi to affect human health. Dermatomycosis and
aspergillosis are two infectious diseases that are caused by molds and A. fumigatus,
respectively (Smith, 1976). Some mold infections, although rarely impacting healthy
individuals, may be fatal for those suffering immunodeficiency, or recovering from burns
or surgery (Kowalski, 2000).
Viable and non-viable fungal particles are associated with health effects (Samson et al.,
1994). Viability is a prerequisite for bioaerosols to be infectious; although it is not a
prerequisite in causing allergenic or toxic effects (Willeke and Baron, 1993). It has also
been reported that fungal spores release allergens during their germination (Mitakakis et
al., 2001; Green et al., 2003). For the above reasons, viable spores are emphasized in this
investigation as more significant than total spore measurements. However, the effects
cause by toxins or beta-glucan are not dependent on spore viability (Bhatnagar et al.,
2005).
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Real-time detection and identification of bioaerosols has become desirable in medical and
agricultural areas, as well as in situations where risk is high. Real-time monitoring has
many advantages over conventional methods, including brief sample detection (in the
order of seconds), reduced labour and greater analytical frequency due to the possibility
of continuous monitoring.
The Ultraviolet Aerodynamic Particle Sizer (UVAPS, model 3312, TSI, St. Paul., MN)
can provide concentrations, size distributions, and fluorescence for particles with
aerodynamic diameters of 0.5 to 15 µm, in real-time. Fluorescence detection is performed
by exciting particles with an UV laser beam at 355 nm and then detecting emissions at
420 to 575 nm.
Viable cells of most organisms, including fungi, have a natural auto-fluorescence due to
biochemical fluorophores such as the reduced fluorescent coenzymes nicotinamide-
adenine dinucleotide (NADH) and nicotinamide-adenine dinucleotide phosphate
(NADPH), the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide
(FAD), and metabolic function riboflavin (vitamin B2) (Li et al., 1991; Brosseau et al.,
2000; Billinton and Knight, 2001). The maximum excitation and emission peaks for the
coenzymes NAD(P)H are 360 nm and 460 nm, respectively (Dellinger et al., 1998;
Billinton and Knight, 2001). Flavins’ maximum excitation peaks are 360 nm and 445 to
470 nm (Dellinger et al., 1998) or 385 nm (Li et al., 1991), with a maximum fluorescence
peak at 530 nm (Billinton and Knight, 2001) or 525 nm as in Li et al. (1991). NAD(P)H
and flavins are largely responsible for fungal fluorescence detected by the UVAPS.
While many studies have been conducted to detect bacteria using the UVAPS (Brosseau
et al., 2000; Agranovski et al., 2003a; Agranovski et al., 2003b), very limited research has
been done on evaluating the method for monitoring airborne fungi. The primary goals of
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this work were to determine the abilities and limitations of the UVAPS with respect to
detection of fungal aerosols. The study was also focused on investigating the possibility
of discriminating between different fungi using the UVAPS technology. It also introduces
a simple and reproducible method for generating airborne fungal spores.
3.2 Materials and methods
All experiments were conducted in a Class II, Type A, Biological Safety Cabinet (SG-400
SterilGARD, E-mail Westinghouse Pty Ltd., Australia) in the International Laboratory for
Air Quality and Health (ILAQH)
3.2.1 The UVAPS calibration The UVAPS is designed to record particles with respect to their fluorescence in channels
1 to 64. The particles with no fluorescent compounds appear in channel 1 and in a very
small fraction in channel 2; while the bioaerosols (with endogenous metabolites) appear
in the channels from 2 to 64, according to their fluorescent intensity. The particles with
higher fluorescent intensity are recorded in the higher channels (TSI-Incorporated, 2000).
The fluorescence spectra detected by the UVAPS are very sensitive to change in the UV
laser pulse energy and photomultiplier tube (PMT) gain, i.e. doubling the quantity of each
of the two parameters would lead to doubling of the measured fluorescence intensity
(TSI-Incorporated, 2000; Agranovski et al., 2003a). In this work, the detected threshold
base line was controlled and checked during the course of the experiments. The UV laser
pulse energy and the PMT gain were set to 50 ± 1% of the laser’s full power and 482 V,
respectively. At such settings, 0.993 µm diameter monodispersed Polystyrene Latex
(PSL) Particles (Duke Scientific Corporation, Palo Alto, CA) started to give weak
fluorescent signals in channel 2, while 0.91 µm diameter Blue Fluorescent (BF)
microspheres (Duke Scientific Corporation, Palo Alto, CA) appeared in the last channel.
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The UVAPS was calibrated using 0.993 µm diameter monodispersed PSL particles and
0.91 µm diameter BF microspheres, which were aerosolized with a Collison nebuliser.
Suspensions were made by diluting one drop from stocks in sterilized, distilled water to
concentrations in the order of 107 particles/mL. Monosized polymer particle suspension,
0.1% solids (SS-2-PXG 0.1%), (SS-5-PXG 0.1%) and (SS-7-PXG 0.1%) standards with
1.05 g/cm3 from DYNO PARTICLES AS were also used for calibration of the UVAPS.
The factory calibration procedure (dry redispersion) was used for these standard particles.
3.2.2 Sample preparation
3.2.2.1 Fungal species used for aerosolization by Collison nebulizer
Penicillium sp. (Australian Collection of Microorganisms - ACM 4616) was inoculated
on Sabouraud Dextrose Agar (SDA) and incubated at 25oC for one week. A suspension of
spore solution was prepared by scraping the surface of the Penicillium mycelia with a
sterilized needle (without touching the agar) and transferring the spores to the Collison
nebulizer jar containing deionized water. Aspergillus niger (American Type Culture
Collection - ATCC 9142) prepared in the same way as Penicillium sp. The measurements
of background concentrations of aerosols generated from distilled water were conducted
before each experiment. In addition, to control the effect of contamination of the samples
with SDA during surface scraping of the mycelia, the blanks of SDA (a few grams in
distilled water) were also tested three times. The measurements of SDA blanks were
conducted in order to evaluate the fluorescent signal of agar, if any, that could contribute
to the total signals when the suspensions of fungal spores were aerosolized.
3.2.2.2 Fungal species used for aerosolization by dry (direct) method
Penicillium sp. (ACM 4616) and A. niger (ATCC 9142) were inoculated on SDA and
incubated at 25oC for one week, then refrigerated for one day before being used in a
direct aerosol generation method. The fluorescent percentage of fungal spores was found
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to decrease as they aged, as well as with an increased frequency of air exposure (Kanaani
et al., 2007), and as such, these samples had a higher fluorescent percentage because they
were young colonies and had not been exposed to air currents. They are subsequently
referred to as having a “high fluorescent percentage”. In addition, cultures with a “low
fluorescent percentage” were used for comparison of the UVAPS performance with the
AGI-30 impinger (as described in section 3.3.3.), which was used as a reference
biosampler. These cultures were obtained as follows. After using the “high fluorescent
percentage” cultures of Penicillium sp. and A. niger in the experiments, the agar plates
with fungal cultures were refrigerated for three months and then used again for generating
fungal aerosols with a “low fluorescent percentage”.
3.2.3 Microscopic analysis A light microscope (Model CX31RTSF, Olympus Corporation, Tokyo, Japan) was used
to investigate the fungal particles. Pieces of transparent adhesive tape were distributed on
the floor of the mixing chamber; while others were placed on the chamber wall, close to
the inlet sampling ports, so that the sticky side was facing towards the released particles
(Figure 3.2). In addition, uncoated microscope slides were distributed on the chamber
floor. After fungal particles were generated, each tape piece was mounted onto a
microscope slide with the sticky side down and viewed together with uncoated slides
under a microscope, using the 40x objective (400x magnification). These tests were
performed three times for both Penicillium sp. and A. niger samples generated by the
direct method.
3.2.4 Experimental set up and procedures
3.2.4.1 Generation of fungal aerosols with a Collison nebulizer
As shown in Figure 3.1, aerosols were generated using a 6-jet Collison nebulizer (BGI
Inc., Waltham, MA), which was operated at a flow rate of 7 L/min. Supply compressed
99
air was filtered with a HEPA filter. Droplets carrying microorganisms were dried by a
silica gel dryer, before entering the mixing chamber and when entering the chamber with
compressed HEPA-filtered air, supplied at a flow rate of 4 L/min. A mixing chamber was
used to provide the same physical sampling points for all tests, to achieve homogenous
particle distribution and to control the concentration of aerosols inside the chamber. The
dimensions of the mixing chamber were 100 ×39 × 39 cm and it was made of aluminium
except for one side, made of Perspex, used as a door.
Air
HEPA Filter DryerFlow Meter
Mixing chamber
HEPA Filter
Exhaust
Biological Safety Cabinet
ba
_ _
UV-APS
Collison nebulizer (c) (c)
Mixing chamber (Box) Fan
Figure 3.1: Experiment set-up (Collison nebulizer method). a, Flow Meter; b, Dryer; (c), Pressure equalizing holes.
3.2.4.2 Dry (direct) spore generation method
In the dry generation method, the fungi were aerosolized directly from the cultures
growing on the agar plates, which were placed inside of the chamber (Figure 3.2). During
experiments, compressed HEPA-filtered air entered the mixing chamber at a flow rate of
10 L/min. The continuous air flow contacted the surface of the Penicillium or A. niger
mycelia at an angle of 60o and a distance of 1.5 cm. Measurements of the total and the
fluorescent particle counts were conducted using the UVAPS.
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Simultaneously, fungal aerosols were sampled with the AGI-30 impingers. The exhaust
airflow from both the UVAPS and the AGI-30 were HEPA-filtered and then returned to
the biosafety cabinet, for safety and to prevent contamination of the experimental area
(Figures 3.1 and 3.2). In all the experiments the UVAPS sample time was 20 s.
Compressed air
Biological safety Cabinet
HEPA Filter
UV-APSAGI-30
Exhaust
Mixing chamber Flow Meter
Petri dish
HEPA Filter
__
(c) (c)
Figure 3.2: Experimental set-up (direct method). (c), Pressure equalizing holes.
3.2.4.3 Operational procedures
The UVAPS operated at 5 L/min and the AGI-30 impinger at 12.5 L/min, while the inlet
air was supplied at 10 L/min. Hence a shortfall of inlet air came from two holes used as
“inlet-outlet equalizing holes”, located on the sides of the chamber (Figure 3.2). As the
chamber was placed inside of the biosafety cabinet, the make-up HEPA-filtered air was
taken directly from the cabinet. The concentrations inside the chamber were measured
prior to each experiment and it was confirmed that the background concentrations were
mostly 0.0 #/cm3. Short inflexible tubing was used on sampling ports to minimise particle
101
losses. The trials in these experiments took place at 23-25oC and 51-60% relative
humidity. This is a moderate humidity range, in which the spores remain as singlets and
do not aggregate (Reponen et al., 1996). Room temperature was used in order to represent
a typical indoor environment and also because temperature plays an insignificant role in
the hygroscopic growth of the particles (Li and Hopke, 1993). To obtain statistically
significant results, the experiments were conducted seven times for each organism.
The AGI-30 impingers with the collection medium were autoclaved before being used in
the tests. The aerosolized fungal spores passed through a jet into a collection medium of
20 ml of 0.1% peptone water with 0.01% Tween 80 and 0.005% antifoam Y-30 emulsion
(SIGMA-ALDRICH, Inc., St. Louis, Mo.). The 0.01% Tween 80 and 0.005% antifoam
were added to improve the impinger efficiency as shown in section 3.3.3. The sampling
times were 10 and 20 minutes for high and low concentration, respectively. After
finishing each experiment, the neck of impinger was flushed with the impinger solution.
The final volume of each impinger was measured and corrected for evaporation. The AGI
samples were brought to a final volume of 100 ml with sterile 0.1% peptone water. Then
0.1, 0.2 and 0.4 ml of each sample were plated onto duplicate SDA plates and then
incubated at 25oC. Aspergillus colonies were counted after 48 hours and Penicillium,
which grows slower, after 60 hours. The concentrations in CFU/m3 were calculated from
the colony numbers, the dilution factor and the volume of sampled air. Blanks were
collected and treated in the same way as fungal samples at each trial, except that the petri
dish was contained SDA only instead of SDA with the interested fungal colonies.
Background concentrations were also measured at each trial with the UVAPS.
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3.3 Results and Discussion
3.3.1 The UVAPS calibration Monodispersed PSL 0.993 µm diameter particles and 0.91 µm diameter BF microspheres
were used at 1.05 g/cm3, and their aerodynamic diameters were 1.02 and 0.93,
respectively (Willeke and Baron, 1993). After these standards were aerosolised with the
Collison nebulizer, the mean aerodynamic diameters were 1.0 µm and 0.90 µm, as
measured by the UVAPS (Figures 3.3b, 3.3c and Table 3.2).
As mentioned above, standard particles of SS-2-PXG 0.1%, SS-5-PXG 0.1% and SS-7-
PXG 0.1% with 1.05 g/cm3 (corresponding to aerodynamic diameters of 2.05, 5.12 and
7.17 µm, respectively) were also used for calibration. Mean aerodynamic diameters of
1.99, 4.98 and 6.98 µm were obtained, which all fell within the diameter accuracy
provided by the supplier (± 3%). The range of the standard particles which were used for
calibration covered the entire size range of fungal aerosols under investigation.
In regard to the fluorescence signals, the UVAPS showed selectivity for the special
materials used in this study. While BF microspheres exhibited fluorescence of 97.6%, the
PSL showed 0.13% (Table 3.2). The normal blank and SDA blank showed a fluorescence
of 0%. The concentrations of BF and SDA aerosols were 22.1 and 18.9 #/cm3,
respectively. The concentration of distilled water blank aerosol generated by Collison
nebulizer was very low (0.13 #/cm3 with mean diameter of 2µm for a 20 sec sampling
time). This concentration was negligible in comparison with the concentration of the
standards.
103
(a)
(b)
Figure 3.3: UVAPS spectra for blank and standards: (a) Blank (direct method). (b) PSL (0.993 µm) generated by Collison nebulizer.
104
(c)
Figure 3.3: UVAPS spectra for Blue fluorescent standards: (c) blue Fluorescent (0.91 µm) generated by Collison nebulizer.
3.3.2 Application of Collison nebulizer for generating fungal aerosols Collison nebulizer was not an effective means for generating fungal aerosols for two
reasons. Firstly, it was unable to generate particles large enough to cover all the fungal
particles under investigation, which can reach up to 6.7 µm in A. niger (see Figure 3.6b),
and hence, a proper comparison between UVAPS and AGI-30 would not be able to be
established. This was confirmed by testing a series of standard particles. While standard
particles of 2 µm in diameter (SS-2-PXG 0.1%) were atomized by the nebuliser and
detected easily (Figure 3.4a), standard particles of 5 µm in diameter (SS-5-PXG 0.1%)
gave no signal using the same method. A mixture of (SS-2-PXG 0.1%), (SS-5-PXG
0.1%), (SS-7-PXG 0.1%), (SS-10-PXG 0.1%) and (SS-15-PXG 0.1%) was also measured
using same method and only a signal for 2 µm was detected (Figure 3.4b). Secondly,
there was a non-homogeneous distribution of fungal particles in the solutions, due to the
hydrophobic nature of the fungi. These two factors contributed to the failure of the
105
instrument in detecting the Aspergillus niger and Penicillium sp. in the proper way.
Although signals were obtained for Penicillium sp. (Figure 3.4c), they were not
reproducible due to the non-continuous spore generation. The Collison nebulizer (BGI
Inc., Waltham, MA) produces aerosols with a mass median diameter of 2 µm and a
geometric standard deviation of 2 (Willeke and Baron, 1993). However there are many
recent studies which produce fungal spores with mean physical dimensions of 1.8, 2.5,
and 3 µm (Grinshpun et al., 2007) or fungal spores with mean aerodynamic diameters up
to 3.1 µm (Yao and Mainelis, 2006) using a 6-jet Collison nebulizer (BGI Inc., Waltham,
MA). More investigations are needed to determine the cut-off size of this nebuliser and its
efficiency with regard to each aerosolised particle size. Since no positive results were
obtained for generating fungal particles by this method, the tests were further conducted
using only the dry (direct) generation method.
106
(a)
(b)
Figure 3.4: UVAPS spectra (APS part) for aerosols generated by Collison nebulizer: (a) (SS-2-PXG 0.1) standard; (b) A mixture of (SS-2-PXG 0.1), (SS-5-PXG 0.1), (SS-7-PXG 0.1), (SS-10-PXG 0.1) and (SS-15-PXG 0.1) standards.
0
1
2
3
4
5
6
7
8
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
Aerodynamic Diameter (µm)
Aerodynamic Diameter (µm)
107
(c)
Figure 3.4: UVAPS spectra (APS part) for aerosols generated by Collison nebulizer: (c) Penicillium species (ACM4616).
3.3.3 Direct generation method
3.3.3.1 Fungal particles morphology
To identify whether the released fungal particles are spores, hyphal fragments or fungal
fragment propagules, the particles were investigated by a light microscope.
Based on the particle characteristics such as shape and size, the particles were classified
as spores (Table 3.1); mostly singlets with a small percentage of spore chains consisting
of two or more spores. Fungal hyphae were not clearly observed on the slides. On the
other hand, similar size distribution and almost the same mean aerodynamic diameters
(Table 3.1) were detected by the UVAPS for all of the samples tested during the course of
these experiments of samples, which indicated the release of fungal spores. The mean
aerodynamic diameters measured with the UVAPS were in an agreement with literature
0
5
10
15
20
25
30
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
Aerodynamic Diameter (µm)
108
data for the size of spores under investigation. It is typical to find most of the airborne
fungi of these sizes in spore form, because spores are dry and easily released by airflow,
especially those at the end of the conidial chains (Ingold, 1979). While hyphal or spore
fragments were not detected at the lower flow rates, at the higher flow rates such as 15,
20, 25 and 30 L/min (corresponding to 5.5, 7.1, 8.5 and 10.2 m/s) fragmentation did
occur; with a fragmentation percentage of 0.98%, 2.7%, 7.9% and 8.1%, respectively.
The fragmentation percentage is defined as the number of samples which contained
fragmented spores as a percentage of the total number of samples at each flow rate.
Table 3-1: Tested fungal characteristics. Fungal Source Conidial Shape Width, Length M.A.Da
spores head colour (µm) Penicillium (ACM 4616) Greenish blue Subgloboseb 2-4c 2.53± 0.10 A. niger (ATCC 9142) Brownish Globose, 2.5-5.0d 3.51 ± 0.08 blackd subglobosed ______________________________________________________________________________________________________ aMean Aerodynamic Diameter as measured by UVAPS. bAs identified by light microscopy. c(Ramirez, 1982) d(Raper et al., 1965)
3.3.3.2 Fungal spore aerosolization
The direct particle generation method showed a distinct difference between the blank and
fungal samples (Figures 3.3a, 3.5a and 3.5b). It has been shown that when the Collison
nebulizer is used for generating bacterial aerosols from non-microbial blanks (e.g., agar
washing, peptone water, broth media), it produces relatively strong fluorescent signals
(Agranovski et al., 2003a). In such cases, the UVAPS is not selective for the specific
microbial fluorescent molecules (Agranovski et al., 2003a). In the dry generation method
of producing fungal aerosols this defect can be avoided, as fungal spores are generated
directly from the agar plates with very little or no effect from the growth medium
(measured as a background). Thus, under controlled conditions, the UVAPS was selective
109
towards the fungal spores under investigation. The fluorescence signals were quite
different for the fungal and the blank aerosols, as well as for the fungal genera examined
(Aspergillus and Penicillium), through the fluorescent percentage and, to a lesser extent,
their sizes (see section 3.3.4.). The concentrations for the blanks (aerosols generated from
sterile agar plates without fungi) were up to 0.1 #/cm3 and the fluorescent percentage was
zero.
The UVAPS determined the abundance and size characteristics of spores for each fungus
tested. As shown in Table 3.1, the fungal spore size characteristics obtained with the
UVAPS were in agreement with the literature data.
For Penicillium sp., 98% of the total fluorescent particles showed fluorescence signals in
the range of 2.1-4.0 µm with the remaining 2% distributed between 1.8-2.0 µm and 4.0-
4.7 µm (Figure 3.6a). For A. niger, 70% of fluorescent particles had aerodynamic
diameters in the range of 2.5-4.1 µm, more than 99% were between 2.3-5.8 µm, while 1%
were detected in the ranges of 2.0-2.2 µm and 5.9-6.7 µm (Figure 3.6b).
These low percentages of large particles (around 6.7 µm for Aspergillus and 4.7µm for
Penicillium) are likely a result of aggregation due to the collision of smaller particles,
which increased at high concentrations, and due to the fact that the minority of particles
were chains of two or more spores.
110
(a)
(b)
Figure 3.5: UVAPS spectra for fungal spores (direct method): (a) Penicillium sp.; (b) A. niger.
111
(c)
Figure 3.5: UVAPS spectra for fungal spores (direct method): (c) Penicillium sp. (low concentration).
112
(a)
(b)
Figure 3.6: UVAPS spectra (APS part) for fungal aerosol: (a) Penicillium sp.; (b) A. niger.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
Aerodynamic Diameter (µm)
Aerodynamic Diameter (µm)
113
(c)
Figure 3.6: UVAPS spectra (APS data) for fungal aerosols: (c) Fragmentation of Penicillium sp. at air flow rate of 25 L/min.
3.3.3.3 UVAPS and AGI 30 correlation
It has been suggested that both the microbial Andersen impactor and the AGI-30
impinger are suitable reference samplers for measuring culturable bioaerosols (Willeke
and Macher, 1999a). The Andersen impactor was not used in this study because the total
concentrations of fungal particles were very high (in the order of 106-107 particles/m3),
which would cause rapid (in less than one minute) overloading of the agar plates (Thorne
et al., 1992). On the other hand, the AGI-30 was reported to be suitable for fungal assays
(Cox and Wathes, 1995). Thorne et al. (1992) found that the AGI-30 is the preferred
sampling method for viable fungi in swine barns where it was reported that Penicillium,
Cladosporium and Aspergillus (which are hydrophobic in nature) are the dominant fungal
species (Chang et al., 2001). In addition, Awad (2002) used the AGI-30 impingers for
sampling viable fungi in the environments where Penicillium, Cladosporium and
Aspergillus species were the dominant fungi. However, Cage et al. (1996) reported that
0
5
10
15
20
25
30
35
40
.5 .7 1 2 3 5 7 10 20
Conc. (dN
#/cm³)
Aerodynamic Diameter (µm)
114
hydrophobic fungal spores may be collected inefficiently, but the addition of surfactant
would improve the capture and retention of these hydrophobic spores. Therefore to
improve the impinger efficiency, the following measures were applied in this study: (1)
sampling times were 10-20 minutes, to minimize the vaporization (the decreasing volume
was 1.1 ± 0.2 ml and 1.9 ± 0.2 ml, respectively) and to decrease particle bounce, the 10-
20 minute sampling time is within the optimal time range for the instrument, so the
impinger bioefficiency for collecting fungal spore is high (Lin and Li, 1998) and although
a longer sample interval would be more representative fungal assay, the physical
collection efficiency for the size range of particles under investigation is significantly
reduced after one hour sampling, due to the evaporation of liquid and to particle
reaerosolization (Grinshpun et al., 1997); (2) Tween 80 (surfactant) was added to the
collection liquid to improve the capture and retention of these hydrophobic spores; (3)
antifoam was added to decrease bubbles and consequently to decrease particles re-
aerosolization; and (4) 20 mL of the collection liquid was used to enhance the sampling
efficiency of the impinger (Cage et al., 1996; Grinshpun et al., 1996).
As shown in Figures 3.7a and b, the UVAPS and the AGI-30 samplers showed a
reasonable correlation for both Penicillium and A. niger (r = 0.911, p < 0.005 and r =
0.882, p < 0.05, respectively). The UVAPS showed a low standard deviation of
fluorescent percentage for both high and low fluorescence cultures (81.5 ± 1.6 and 25.1 ±
0.5% for Penicillium and 62.4 ± 1.1 and 15.3 ± 0.8% for A. niger) compared to the
culturable percentage obtained by the AGI-30 (67.25 ± 15.5 and 22.0 ± 2.5% for
Penicillium and 50.5 ± 14.0 and 16.8 ± 1.8% for A. niger). The concentrations obtained
with the AGI-30 were smaller than those by the UVAPS, which may be due to the
hydrophobic nature, culturability and viability of the spores under investigation (since the
UVAPS is measuring viable spores whereas the results from AGI-30 are culturable
115
spores). There was one exception when the results of the AGI-30 (for A. niger) were
higher than for the UVAPS (16.8 and 15.3%, respectively). This may be explained by the
separation of cluster spores from single spores in the collection fluid of the AGI-30
(Willeke and Macher, 1999a); however, this issue needs more investigation. The
concentrations of total particles generated during the experiments were 8-16 #/cm3 for
Penicillium and 4-13 #/cm3 for A. niger for a high fluorescent percentage samples, and
around 2 and 3 #/cm3 for Penicillium and A. niger for low fluorescent percentage
samples, respectively. The culturable percentage of blanks was zero for both Penicillium
and A. niger in all trials.
116
(a)
R2 = 0.8299
0
20
40
60
80
100
0 20 40 60 80 100
Fluorescent %, UVAPS
Cul
tura
ble
%, A
GI-3
0
(b)
R2 = 0.7773
0
20
40
60
80
0 20 40 60 80
Fluorescent %, UVAPS
Cul
tura
ble
%, A
GI-3
0
Figure 3.7: Fungal spore measurements of UVAPS and AGI-30 impingers during the experiment investigating their correlation: (a) Penicillium sp.; (b) A. niger.
Such correlation may be due to the UVAPS and the AGI-30 impinger both collecting
aerosols from the same zone (sampling ports were in close proximity). Figure 3.8 also
shows a linear relationship between the total and fluorescent particle concentrations. The
high linearity shows the homogenous distribution of fluorescent biomolecules over the
population of fungal spores. Table 3.2 also shows that the UVAPS behaved in a selective
manner towards the fungal spores. The fluorescence signals of both species may be due to
their bimolecular (NADH, NADPH and riboflavin) content.
117
Figure 3.8: The range of total and fluorescent particle concentrations from fungi as measured by UVAPS during the experiment investigating the correlation with AGI-30 impingers. Table 3-2: Aerosol results as measured by UVAPS and impingers. Aerosol
Total particlesa
Fluorescent particlesb
Culturable particlesc
Conc. M.A.Dd G.S.De Conc. M.A.D F%f Conc. Cul. g %
#/m3 µm #/m3 µm CFU/m3
Blank 9.0E+04 0.86 1.29 0.0 0.0 0.0 0.0 0.0
PSL (0.993) 1.8E+07 1.00 1.21 2.4E+04 1.40 0.13 - -
Bf (0.91) 1.6E+07 0.90 1.15 1.6E+07 0.90 97.6 - -
Penicillium 1.2E+07 2.55 1.28 9.8E+06 2.50 ± 0.11 81.5 8.0+E06 67.3
Penicilliumh 3.0E+06 2.66 1.24 7.5E+05 2.63 ± 0.10 25.1 6.6+E05 22.0
A. niger 5.5E+06 3.47 1.31 3.4E+06 3.47 ± 0.09 62.4 2.8+E06 50.5
A. nigeri 1.9E+06 3.54 1.26 2.9E+05 3.53 ± 0.07 15.3 3.2+E05 16.8
a Total particles were particles counted through the channels 1-64. b Fluorescent particles were counted through the channels 2-64. c Culturable particles were measured by AGI-30 impingers; cultured on Sabouraud Dextrose Agar plates. d M.A.D is the mean aerodynamic diameter. e G.S.D is the geometric standard deviation. f F% is the fluorescent percentage. g Cul.% is the percentage of culturable particles. h Penicillium sp. with low fluorescence culture. i A. niger with low fluorescence culture.
118
3.3.3.4 The potential of the UVAPS in differentiating between fungal species
All fungal spores contain the same sources of autofluorescence such as NADH, NADPH
and riboflavin (Li et al., 1991; Brosseau et al., 2000; Billinton and Knight, 2001).
However, the fluorescence signals of these biomolecules may vary due to their
concentrations and the environmental conditions under which the fungal colonies are
placed.
For high fluorescence cultures, the average percentage fluorescence for Penicillium was
81.5 ± 1.6% and for A. niger the average was 62.4 ± 1.1%; while for low fluorescence
cultures, the percentage was 25.1 ± 0.5 % for Penicillium and 15.3 ± 0.8% for A. niger
(Figure 3.9 and Table 3.2). This suggests that the spores of Penicillium species have more
riboflavin and/or NAD(P)H than those of A. niger, as both were exposed to the same
culturing and test conditions. It was also found that the fluorescent percentage of spores
increases with increasing aerodynamic diameter within the species (Kanaani et al., 2007),
which indicates that larger particles are metabolically more active. On the other hand,
Penicillium aerosols, which had a mean spore size smaller than that for Aspergillus
aerosols, had larger fluorescent percentage than Aspergillus aerosols. This may suggest a
difference in the fluorescent properties (e.g. amount of fluorophores) of viable spores for
these two species or that there is a difference in the fraction of viable spores between
these two species. It may also be due to other reasons which need further investigation. In
regard to the spore size, although the mean aerodynamic diameters of these fungi were
close to each other, they were not identical: 2.53± 0.10 μm for Penicillium sp. and 3.51 ±
0.08 μm for A. niger (Tables 3.1 and 3.2). These two aerosols also differed in their size
distribution range (Figure 3.6a and b). In the entire study (which investigated thousands
of samples), not one spectra for the Penicillium samples was similar to the Aspergillus
samples.
119
The UVAPS has demonstrated the ability to sort fungi (Penicillium and A. niger) by
fluorescence percentage and size when cultures were exposed to the same growth
conditions. On the other hand, it has to be noted that the UVAPS would not sort these two
fungi in the field (ambient air samples). It has been shown that the percentage
fluorescence of A. niger and Penicillium sp. decreases with the spore age and frequency
of air exposure (Kanaani et al., 2007). Huber et al.(2000) also reported that the
fluorescence spectra for free NADH and those bound to protein are different. So in the
field, where microorganisms are subjected to different environmental conditions (such as
air speed) and due to the presence of a mixture of microorganisms of varying ages, the
fluorescence signals would not be useful in differentiating between fungal spores. In
addition, the presence of non-biological aerosols, which may also fluoresce at the same
wave length (355nm) as spores, will add to the difficulties of differentiating
microorganisms in environmental samples.
020406080
100
0 5 10 15 20
Total particle concentrations, #/cm3
Fluo
rese
nt %
AspergillusPenicillium
Figure 3.9: The range of total concentrations and fluorescent percentage for high fluorescent percentage cultures as measured by UVAPS, during the experiment investigating the correlation with AGI-30 impingers.
3.3.3.5 The relationships between total and fluorescent particle concentrations
The total and the fluorescent particle concentrations measured by the UVAPS showed a
linear relationship until the concentrations of total particles reached 70 particles/cm3 for
Aspergillus niger Penicillium sp
120
both species (Figure 3.10). Beyond this point, the graph ceases to be linear and total
particle number increases at a greater rate than fluorescent particle number. This can be
explained by considering the UVAPS as two distinct parts, the APS part (for counting the
total particles) and the UV laser part (for counting the fluorescent particles). When a
particle passes through two constant red laser beams, the scattered light is collected on the
photodiode detector and the APS part counts one particle. The time needed for the APS to
count another particle is less than that needed for the UV laser to fire its pulse (the dead-
time for the UV laser is 200 µs). So at high particle concentrations, when two or more
particles enter the UV detection zone, the UV beam during the 200 µs dead-time, will
illuminate only one particle to be detected by the photomultiplier tube detector and hence
the UV laser counter misses the other particles which already have been counted by the
APS (Agranovski et al., 2003a). Further, the particles can also be counted coincidently by
the APS part, so that when more than one particle is present in the detection zone (as will
often occur with increasing particle concentrations), all of the particles in the detection
zone will be counted, however this is not the case with the UV laser reading (TSI-
Incorporated, 2000). This study showed that the maximum detection limit of the
fluorescent particles for the UVAPS (the linear region illustrated in Figure 3.10) is
approximately 7 × 107 particles/m3 (for the total particles). In other words, if the
fluorescent percentage of the sample is 100%, then the efficiency of the UVAPS for
measuring fluorescent particles will be approximately 7 × 107 particles/m3. This result is
consistent with the upper detection limit of the UVAPS for measuring bacteria, which is 6
× 107 particles/m3 (Agranovski et al., 2003a).
121
020406080
100120
0 50 100 150 200
Total particles concentration, #/cm3
Fluo
rese
nt p
artic
les
con
cent
ratio
n, #
/cm
3
Figure 3.10: Fluorescent particles of Penicillium sp. as a function of total particles by UVAPS, similar trend was obtained for A. niger (data not shown)
On the other hand, the sensitivity of the UVAPS was high with respect to the fungal
spores under investigation. The instrument showed a good ability to count and to
differentiate A. niger and Penicillium sp. spores under controlled laboratory conditions.
When measuring one spore, the UVAPS locates it in one of the 64 channels: in channel
one (if the particle is non-viable or when the content of specific fluorophores is lower
than the detection limit of the UVAPS) or in one of the fluorescent channels (2-64),
depending on the amount of fluorophores in the particle. While Figure 3.5c shows fungal
spores of Penicillium sp. with a concentration of 0.006 particles/cm3, the UVAPS lower
detection limit was found to be 0.01 particles/cm3 (as total concentration). Some of the
spores are located in the fluorescent channels, with one in channel one. The sensitivity of
the UVAPS in measuring fungal spores depends not only on the spores themselves, but
also the instrument setting parameters. While the ability of the UVAPS for measuring
fungal spores was high, it was limited to measuring only individual bacterial spores
(Agranovski et al., 2003a) when similar UV laser pulse energy and the PMT gain settings
were used (50 ± 1% of the laser’s full power and 482 V in this study and 50% and 500 V
for Agranovski et al., 2003). For instance, in Agranovski et al. (2003a) the fluorescent
percentage of B. subtilis spores was 1.5 %, which was less than that of background
122
aerosols (1.8 %), compared to 81.5% and 62.4% for Penicillium and A. niger,
respectively. Again, this difference may be due to the amount of viable spores for the two
microorganisms (fungi and bacteria) under investigation and also may be due to the
smaller size of B. subtilis (count median diameter of 1.09 μm) compared to fungal spores.
It has to be, however, noted that the concentration of fungal spores in the environmental
samples are typically considerably smaller than what was investigated in this study. For
example, (Hargreaves et al., 2003c) has reported that the concentrations of airborne fungi
in environmental samples measured outdoors and indoors (in 14 residential suburban
houses in Brisbane) are 1133 ± 759 CFU/m3 and 810 ± 389 CFU/m3, respectively. The
study by Lee et al. (2006) has reported that the concentration of airborne fungi in six
Cincinnati homes is typically between 0 and 1362 CFU/m3. As UVAPS lower detection
limit was found to be 0.01 particles/cm3, to study fungal aerosols at such concentration
levels, the UVAPS has to be operated with a dedicated concentrator. The sensitivity of the
instrument in the field requires, however, further investigation.
3.4 Conclusions
This work has investigated the performance of the UVAPS for monitoring fungal aerosols
under controlled laboratory conditions. The instrument was found to be quite sensitive for
detecting the fluorescent biomolecules present in the fungal spores investigated. The
UVAPS results were comparable with the results of the AGI-30 impinger, which was
used as a reference sampler. A linear relationship was observed for the total particles and
the fluorescent particle concentrations, measured by the UVAPS within the concentration
range up to approximately 7 × 107 particles/m3. This number represents the upper limit of
detection of the UVAPS for fungal particles.
123
The study has also investigated two methods for generating fungal aerosols. While the
Collison nebulizer was found to be unable to generate fungal aerosols with diameters of 5
µm and larger, the dry generation method has proved to be reproducible and easy to
control, as well as simple and inexpensive.
Acknowledgements
This project was supported by the grant 1-9311-8174 from the Research Triangle
Institute, PO Box 12194 Research Triangle Park, NC 27709-2194. Special thanks go to
Dr David Ensor, Aerosol Science and Nanotechnology, whose vision made this study
possible. Support and assistance provided by ILAQH staff is gratefully acknowledged.
124
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of relative humidity on the aerodynamic diameter and respiratory deposition of
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Hoekstra, E. S. (Eds.). (1994). Health implications of fungi in indoor
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bioaerosol sampling methods in barns housing swine. Applied and Environmental
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TSI-Incorporated. (2000). Ultraviolet Aerodynamic Particle Sizer Spectrometer, Model
3314. Instruction Manual.
Verhoeff, A. P. and Burge, H. A. (1997). Health Risk Assessment of Fungi in Home
Environments. Annals of Allergy, Asthma and Immunology, 78, 544-556.
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Lockey, R. F. and Bukantz, S. C. (Eds.), Allergens and Allergen Immunotherapy.
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and applications. John Wiley & Sons, Inc, New York.
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CHAPTER 4 : Performance assessment of UVAPS: Influence of fungal spore age and air exposure
Hussein Kanaania, Megan Hargreavesa, b, Zoran Ristovskia and Lidia
Morawskaa,
aInternational Laboratory for Air Quality and Health, Queensland University of
Technology, Brisbane, QLD, Australia
bSchool of Life Sciences, Microbiology Section, Queensland University of
Technology, Brisbane, QLD, Australia
(2007) Journal of Aerosol Science 38: 83-96
129
Statement of Joint Authorship
Title: Performance assessment of UVAPS: Influence of fungal spore age and air
exposure
Authors: Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia
Morawska
Hussein Kanaani: came up with the original idea; developed experiment
design; conducted experiments; analysed data and wrote the paper.
Megan Hargreaves: contributed to experimental design, provided technical
help with microbiological techniques and offered editorial comments throughout
manuscript writing.
Zoran Ristovski: contributed to data analysis, provided technical assistance,
helped in solving problems during the experiment and provided editorial
comments during manuscript writing.
Lidia Morawska: contributed to experimental design, provided criticism and
advice, helped in solving problems during the experiment, offered editorial
comments throughout the preparation of the manuscript.
130
Abstract
This work focused on two main outcomes. The first was the assessment of the
response of the Ultraviolet Aerodynamic Particle Sizer Spectrometer (UVAPS)
for two different fungal spore species. The UVAPS response was investigated as
a function of fungal age and the frequency of air current that their colonies
exposure to. This outcome was achieved through the measurement of fungal
spore fluorescent percentage and fluorescent intensity throughout a period of
culturing time (three weeks), and the study of their fluorescent percentage as a
function of exposure to air currents. The second objective was to investigate the
change of fungal spore size during this period, which may be of use as a co-
factor in this differentiation. Fungal spores were released by blowing the
surface of the culture colonies with continuous filtered flow air. The UVAPS
was used to detect and measure auto-fluorescing biomolecules such as
riboflavin and nicotinamide adenine dinucleotide phosphate (NAD(P)H) present
in the released fungal spores.
The study demonstrated an increase in aerodynamic diameter for fungal spores
under investigation (Aspergillus niger and Penicillium species) over a period of
time. The fluorescent percentage of spores was found to decrease for both
fungal genera as they aged. It was also found that the fluorescent percentage for
tested fungi decreased with frequency of air exposure. The results showed that,
while the UVAPS could discriminate between Aspergillus and Penicillium
species under well-controlled laboratory conditions, it is unlikely to be able to
do so in the field.
Keywords: fungal spores, culturing time, fluorescent percentage, spore size
131
4.1 Introduction
The presence of biological aerosols such as bacteria and fungi in the air is
associated with risk to human health (Murray et al., 1990; Vijay et al., 1999b;
Maus et al., 2001). In particular, fungi are responsible for many allergic diseases
such as asthma, allergic bronchopulmonary mycoses and hypersensitivity
pneumonitis, to name just a few (Vijay et al., 1999b). To assess and minimise
risks to human health, it is important to develop a better understanding of the
dynamics of these aerosols in the air, which can only be achieved with the
application of fast and accurate real-time detection methods.
Traditional methods used for detection of airborne biological aerosols are based
on sample collection and subsequent laboratory analysis. As such, they only
allow for snapshots of biological particle characteristics and are unable to yield
high resolution time series of the characteristics. This is a major limitation,
preventing progress on the understanding the dynamics of these particles. As
such, further work aimed at extending the capabilities of real-time detection
techniques, is very important for developing a better understanding of the
science of biological aerosols and without developing these techniques, it would
be difficult to achieve any real progress.
During the last decade, different trials with instruments for measurement of
fluorescence spectra as a method for real-time detection of single viable
airborne bioaerosols have been reported. These can be divided into three groups.
The first group includes trials and studies to design and test an instrument
capable of differentiating between biological and non biological aerosols such
as a Fluorescence Spectrum Analyser and an Ultraviolet Aerodynamic Particle
132
Sizer (UVAPS) (Hill et al., 1995; Pinnick et al., 1995; Chen et al., 1996;
Nachman et al., 1996; Hariston et al., 1997; Pinnick et al., 1998; Ho et al., 1999;
Brosseau et al., 2000; Kaye et al., 2000; Pan et al., 2003). The second group of
studies aimed at designing and testing an instrument with the capability to
characterise particle composition in order to discriminate between the
bioaerosols themselves (Cheng et al., 1999; Pan et al., 1999; Seaver et al., 1999;
Weichert et al., 2002; Sivaprakasam et al., 2004). Some of these studies used
multiple UV excitation wavelength to create more than one fluorescence spectra
for each species under investigation (Cheng et al., 1999; Sivaprakasam et al.,
2004). The third group coupled the UVAPS with other technologies, such as
wet chemistry technology, so that if unusual aerosols were detected by UVAPS,
samples were collected for further analysis to identify the microorganisms using
the wet chemistry technique (Ho, 2002). The key findings for the first group are
most encouraging; however groups two and three still require further
investigation.
The UVAPS operation is based on the excitation and emission of auto-
fluorescent biomolecules, which exist in most bioaerosols. The main
biomolecules present in fungal spores are reduced fluorescent coenzymes:
nicotinamide-adenine dinucleotide (NADH), nicotinamide-adenine dinucleotide
phosphate (NADPH), and riboflavin (Li et al., 1991; Brosseau et al., 2000;
Billinton and Knight, 2001). However, the basis of the instrument’s operation,
the fluorescence of the excited biomolecules, has been found to be strongly
affected by environmental and biomolecule-related factors. For example,
Agranovski et al. (2003b) have shown that bacterial stress has an impact on
these fluorescence properties. Huber et al. (2000) found that the fluorescence
133
spectra for free NADH and those bounded to protein were different. Schmit and
Brody (1976) found that Neurospora crassa spores had low level of reduced
cofactors, NADH and NADPH, compared with a high level for its mycelia. It
was also shown that many non biological aerosols such as peptone water, broth
or other materials, had strong fluorescent signals (Agranovski et al., 2003a).
Thus there is a need for a better understanding of these environmental factors
and their effect on the instruments response to different bioaerosols.
It can be seen from the above summary that the UVAPS has been the main
research tool for real-time detection of viable bioaerosols to date. Whilst a
significant amount of work has been published regarding the application of the
UVAPS to the aerosols carrying bacteria (Agranovski et al., 2003a; Agranovski
et al., 2003b), only a very limited amount of work has been done on the
fluorescence spectra of fungal spores and application of the UVAPS to detection
of fungal spores present in the air. Work by Kanaani et al.(2006), addressed the
efficiency and the limits of the UVAPS in detecting fungal spores, showing the
ability of the instrument in detecting and measuring fungal spores and that its
upper detection-limit (the point beyond which the relationship between total and
fluorescent particle concentration cease to be linear) of fluorescent particles was
around 7 × 107 particles/m3. However, there is still a need for further
characterisation and validation of the instrument, and more research is necessary
to develop a full understanding of the capabilities and limitations its application,
for studies of airborne fungal spores.
The aim of this work was to characterise and quantify the effects of aging of
fungal spores and of repeated air exposure, on the ability of the UVAPS to
134
discriminate between different species. The fungi included in the study were
Aspergillus niger and Penicillium species. The novel approach used in the study
measured relative fluorescence intensities and, to some extent, spore size
distribution, utilising the UVAPS for these two parameters for the first time, in
order to investigate its potential to discriminate between fungal spores.
4.2 Materials and Methods
The work was conducted at the International Laboratory for Air Quality and
Health (ILAQH) at Queensland University of Technology, inside a Class II,
Type A, Biological Safety Cabinet (SG-400 SterilGARD, E-mail Westinghouse
Pty Ltd., Australia).
Three sets of experiments were conducted using the UVAPS. The first
experiment was to investigate the effects of fungal spore age on their size and
fluorescent percentage, while the second experiment studied fungal spore
fluorescent percentage as a function of the frequency of air exposure. The third
experiment identified whether the particles released were spores or not. After
the fungal species were cultured, they were released into a purpose-built box
and their fluorescence signals and size distributions were measured using the
UVAPS.
4.2.1 Aerosol preparation
Penicillium (ACM 4616) was inoculated onto three Sabouraud Dextrose Agar
plates (SDA), and incubated at 25 oC for a total of twenty-one days. The
incubated cultures were tested after two, four, seven, fourteen and twenty-one
days. The same steps were followed in preparing Aspergillus niger (ATCC
9142).
135
Another three plates of each species were incubated for seven days, released in
the box and sampled by the UVAPS; then refrigerated for a further week,
sampled again and later refrigerated for another week to be used a third time as
discussed in section 2.2.3.
To minimize the impact of the variation in culture characteristics during fungal
growth, which occurs commonly (Raper et al., 1965), the following protocol in
culturing was applied. Firstly, a single culture divided into five segments was
used instead of five separate cultures, and secondly, the replicate cultures were
prepared using the same conditions as the original culture.
4.2.2 Apparatus description The Ultraviolet Aerodynamic Particle Sizer, [UVAPS, model 3312, TSI, St.
Paul., MN,] is the instrument which was designed to monitor and detect
bioaerosols. It provides accurate particle count size distributions, as well as a
real-time fluorescence for particles with aerodynamic diameters of 0.5-15 µm.
Fluorescence measurements are produced by exciting particles with an UV laser
beam at a wavelength of 355 nm and then detecting the fluorescence emission
from 420 to 575 nm.
The fluorescence spectra detected by the UVAPS are very sensitive to change in
the UV laser pulse energy and photomultiplier tube (PMT) gain, i.e. doubling
the value of each of the two parameters would lead to doubling of the measured
fluorescence intensity (TSI-Incorporated, 2000; Agranovski et al., 2003a). The
detected threshold baseline was controlled and checked during the course of this
study. The UV laser pulse energy was set to 50 ± 1% of the lasers full power
and 482V were applied to the PMT to produce a measurable gain.
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Using the UVAPS, aerosols with no fluorescent compounds appear in channel 1
and in a very small fraction at channel 2; while the bioaerosols (with
endogenous metabolites) appear in the channels from 2 to 64. The particles with
higher fluorescent intensity will be found at higher channels (TSI-Incorporated,
2000).
The mixing chamber in this study, used to provide homogenous particle
distribution before sampling to the UVAPS, was made of aluminium with one
side made of Perspex used as a door. Its dimensions were
(100cm×39cm×39cm).
4.2.3 Experimental Methodology The instrument calibration and background measurements were monitored
before and after each experiment conducted in this study. The average
background was subtracted from each single reading before data interpretation.
However, in most cases it was found to be negligible compared to the tested
sample. In all these experiments the UVAPS sample time was 20 seconds. Each
data point presented in this paper is a mean value of at least three replicate
measurements.
The experiments were conducted at temperatures inside the box ranging from 22
to 26 oC and relative humidity of 50 to 54%. This is a moderate humidity range,
in which the spores remain as singlets and do not aggregate (Reponen et al.,
1996). In contrast to the humidity, temperature plays an insignificant role in the
hygroscopic growth of the particles (Li and Hopke, 1993), therefore room
temperature was used for convenience and to represent typical indoor
environment.
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4.2.3.1 UVAPS calibration.
The UVAPS was calibrated using 0.993 µm particle diameter monodisperse
Polystyrene Latex (PSL) Particles (Duke Scientific Corporation, Palo Alto, CA)
and 0.91 µm particle diameter Blue Fluorescent (BF) microspheres (Duke
Scientific Corporation, Palo Alto, CA). Both were used at a density of 1.05
g/cm3 and their aerodynamic diameters (diameter of unit-density sphere) were
1.02 and 0.93 µm, respectively (Willeke and Baron, 1993). Suspension of each
was made by diluting one drop from stock in sterilized distilled water to a
concentration of the order of 107 particle/m3.
PSL aerosols were generated using a 6-jet Collison nebulizer (BGI Inc.,
Waltham, MA). The nebulizer was operated at a flow rate of 7 L/min and the
supply compressed air was filtered with a HEPA filter. Droplets carrying
aerosols were dried before entering the mixing chamber by a silica gel dryer,
and in addition, when entering the chamber, by compressed-HEPA-filtered-air.
A schematic representation of the experimental set-up is presented in Figure 4.1.
Figure 4.1: Experimental set-up for the UVAPS calibration, a, Flow Meter; b, Dryer; (c), Pressure equalizing holes.
Air
HEPA Filter Dryer Flow Meter
Mixing h b
HEPA Filter
Exhaust
Biological Safety Cabinet
ba
__
UV-APS
Collison nebulizer (c) (c)
Mixing chamber (Box)
138
Monodispersed polymer standard particle suspensions, from DYNO
PARTICLES AS, of 0.1% solids (SS-2-PXG 0.1%), (SS-5-PXG 0.1%) and (SS-
7-PXG 0.1%) and of the density of 1.05 g/cm3, corresponding to particle
aerodynamic diameters of 2.05, 5.12 and 7.17 µm respectively, were also used
for calibration of the UVAPS. Procedure as recommended by the UVAPS
manufacturer (TSI) was followed in the instrument calibration, and it was
conducted before and after each experiment.
The mean aerodynamic diameters of standards obtained were within the
diameter accuracy provided by the supplier (± 3%). The size range of standard
particles that were used for calibration covers the size of fungal spores under
investigation.
4.2.3.2 The fungal spore size and fluorescent percentage as a function of fungal age
The experiments were conducted using the set-up shown in Figure 4.2. The dry
generation method, which was described in detail by Kanaani et al (2006), was
used for fungal spore release. In this method, the compressed air was filtered by
a HEPA filter and introduced into the mixing chamber at a flow rate of 10
L/min in all the cases, except for the two-day-old cultures for which a flow rate
of 20 L/min was used. Spore release was induced by directing a narrow jet of air
at the surface containing Aspergillus or Penicillium mycelia at an angle of 60o
from a distance of 1.5 cm. This short distance above the spores was used so it
would not affect the adjacent sections. The culture dish was divided into
approximately five equal sections by marking the dish bottom using a narrow
paper tape and pen. Each marked section was used for experiments applying a
specific culture time, i.e. two, four, seven, fourteen or twenty-one days. The
139
exhaust airflow of the UVAPS was HEPA- filtered and the airflow was returned
to the biological cabinet to prevent any contamination (Figure 4.2).
Figure 4.2: Experimental set-up (direct method generation), (c) Pressure equalizing holes.
Sampling by the UVAPS was conducted from the same sampling point within
the chamber during all the experiments to measure spore concentration,
fluorescent particle counts and total particle counts.
4.2.3.3 Fungal spore fluorescent percentage as a function of the frequency of exposure
The same method of spore release was used as described above. The culture
dish was divided into three, approximately equal, spore covered sections. After
the first seven days of culturing time, measurements were conducted for one
section only. The airflow was directed towards each individual section via
specific exposure points and the air speed decreased as the distance from the
Compressed air
Biological safety Cabinet
HEPA Filter
UV-APS
Exhaust
Mixing chamber Flow Meter
Petri dish
HEPA Filter
__
(c) (c)
140
exposure point increased. As such, it was at a minimum when it reached the
adjacent section, so the effect on the adjacent section was negligible. This
section was exposed to the air current at a flow rate of 10 L/min for 20 minutes,
during which the sampling from the chamber was conducted. The culture was
then refrigerated for seven days and the measurements were repeated for the
previously exposed section, as well as conducted for the second section. After
the culture had been left in the refrigerator for another seven days,
measurements of fungal spores from all the three sections were conducted.
Hence the last (third) section was exposed to compressed air once, the second
section was exposed twice and the first section underwent three exposures. The
experiment was repeated three times with three cultures exposed to same
conditions.
4.2.3.4 Fungal particle identification
In parallel to culture testing, the released fungal particles were identified, using
an optical microscope, to determine whether they were in fact spores or not.
Pieces of transparent adhesive tape and uncoated microscope slides were placed
inside the box, so released particles attach to or fall on them. This was followed
by microscopy testing to identify the releasing particles, as described in detail
by Kanaani et al. (2006). A light microscope (Model CX31RTSF, Olympus
Corporation, Tokyo, Japan) was used to identify the particles.
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4.3 Results
4.3.1 Fungal particle identification Using the optical microscope and based upon particle characteristics such as
shape, size and appearance, the particles were identified as spores. However,
fungal hypha was not recognized on these slides. Using the UVAPS, the mean
aerodynamic diameter for Aspergillus and Penicillium fungal particles at age
one and two weeks were 2.40± 0.12 and 3.55± 0.14, respectively. The mean
aerodynamic diameters results that obtained by UVAPS for Aspergillus and
Penicillium were in good agreement with literature data for the spores under
investigation (Raper et al., 1965; Ramirez, 1982). The above results support
each other and indicate that the sampled particles were fungal spores, for detail
see Kanaani et al.(2006) and the following section in this study.
4.3.2 Fungal spore size It can be seen from Figure 4.3a and Table 4.1a that the mode aerodynamic
diameter of Aspergillus spores was 3.05µm for two-, four- and seven-day-old
spores. By the 14 and 21 day of culturing time, the mode increased to reach 3.28
and 3.52 µm, respectively. The percentage of spores in the large size range
(beyond the mode, i.e. 3.52-6.24 µm) also increased (the spectrum shifted to the
right), whilst spores in the small size range (lower than 3.28 µm) decreased
(Table 4.1a).
Figure 4.3b and Table 4.1b show that the spores of Penicillium follow the same
trend as that of the Aspergillus. While the spore mode was 2.29 µm for two-,
four- and seven-day-old spores, it increased to 2.46 µm and 2.64 µm for the 14-
and the 21-day old spores, respectively. The percentage of spores in the large
142
size range (beyond 2.46 µm) increased with age, while spore size lower than
2.46 µm decreased (Table 4.1b).
(a)
0
5
10
15
20
25
1 10Spore aerodynamic diameter, µm
Spo
re c
once
ntra
tion,
% 2 days
4 days
7 days
14 days
21 days
(b)
0
5
10
15
20
25
30
1 10Spore aerodynamic diameter, µm
Spo
re c
once
ntra
tion,
%
2 days
4 days
7 days
14 days
21 days
(c)
0
5
10
15
20
25
1.00 10.00Spore aerodynamic diameter, µm
Spo
re c
once
ntra
tion,
%
Penicillium 4 days
Penicilium 21 days
Aspergillus 4 days
Aspergillus 21 days
Figure 4.3: Average spore size distribution spectra as a function of culturing time: (a) Aspergillus; (b) Penicillium; (c) Comparison of Aspergillus and Penicillium.
143
Table 4-1: Average percentage of different sizes of fungal spores at different ages: (a) Aspergillus; (b) Penicillium. (a)
aDi. Time
(days) 2.29 2.46 2.64 2.84 3.05 3.28 3.52 3.79 4.07 4.37 4.7 5.05 5.43 5.83
2 1.74 2.24 3.44 14.6 23.1 20.9 19.5 4.88 5.76 2.44 1.44 0 0 0
4 0.29 2.45 10.8 19.3 21.1 15.4 14.2 10.7 4.07 0.73 0.33 0.12 0.22 0.2
7 0.43 1.64 8.58 15.1 20.5 14.8 16.6 12.9 6.01 2.23 0.32 0.32 0.24 0.16
14 0.31 0.95 3.59 9.8 18.4 18.8 14.6 14.2 10.3 5.16 2.23 0.73 0.45 0.24
21 0.10 0.2 0.62 3.32 11.3 18.4 18.9 16.5 13.8 9.87 4.7 1.34 0.47 0.28
(b) aDi. Time
(days) 1.60 1.72 1.84 1.98 2.13 2.29 2,46 2.64 2.84 3.05 3.28 3.52 3.79 4.07
2 0.60 1.81 2.72 9.67 19.9 24.5 21.5 13 3.93 1.21 0.3 0 0.6 0.3
4 0.08 0.2 1.07 6.49 20 22.8 17.4 16.7 9.09 3.47 1.34 0.51 0.41 0.43
7 0.11 0.25 1.1 5.68 16.3 21.7 18.3 17.9 10.5 4.89 1.62 0.77 0.45 0.39
14 0.13 0.08 0.31 1.84 7.41 19.7 22.4 21.2 16.1 7.22 2.39 0.59 0.43 0.26
21 0.04 0.06 0.25 1.23 4.84 14.5 20.1 22.2 19.5 11.2 3.54 1.38 0.63 0.45
Decreasing with age Increasing with age aAerodynamic diameter in µm.
144
Spore sizes of Aspergillus and Penicillium are cited in literatures as falling in the ranges
from 2.5 to 5.0 µm (Raper et al., 1965) and 2 to 4 µm (Ramirez, 1982), respectively. The
results of this study are in agreement with these figures.
4.3.3 Fungal spore fluorescence as a function of culturing time Total particle number is the number of particles counted by the UVAPS in the channels 1-
64, while fluorescent particle number is the number of particles counted in the channels
2-64. Fluorescent percentage is the number of fluorescent particles as a percentage of
total particle number.
Figure 4.4 shows that total fluorescent percentage decreases with increasing age of the
spores for both Aspergillus and Penicillium. The total fluorescent percentage of
Penicillium is higher than that of Aspergillus for each age of the spores.
50
60
70
80
90
100
2 4 7 14 21Culturing time, days
Fluo
resc
ent P
erce
ntag
e, %
AspergillusPenicillium
Figure 4.4: Fluorescent percentage as a function of culturing time of Aspergillus and Penicillium.
The results also show similar trends for spore size and its fluorescent percentage for both
Aspergillus and Penicillium species (Table 4.2), i.e. fluorescent percentage of spores
increase with increasing aerodynamic diameter. As presented in Table 4.2, the fluorescent
145
percentage for Aspergillus spores with diameters of 2.64 µm and 4.70 µm, were 48.1 ±
13.7 and 93.3 ± 5.0, respectively. Alternatively, Penicillium spores showed a fluorescent
percentage of 58.9 ± 14.0 and 97.8 ± 2.0, for diameters of 1.84µm and 3.05µm,
respectively. These measurements were conducted using spore samples obtained after
seven days of culturing. A similar trend was found for the other ages.
4.3.4 Fluorescent Intensity The intensity of spore fluorescence was investigated in this work by two different
methods. Firstly, investigation was conducted in terms of the spore concentration
percentage (for all diameters in a single channel) versus fluorescent intensity, which is
proportional to the channel number. Spore concentration percentage is the ratio of the
sum of spore concentration of all diameters with same fluorescent intensity, and therefore
the same fluorescent channel (single channel) to the sum of spore concentration of all
diameters in all 64 channels expressed in percentage. The fluorescent intensity
distributions are shown in Figure 4.5. The fluorescence intensity distribution of 0.993 µm
monodispersed PSL Particles was markedly different to that of 0.91 µm BF microspheres.
The non fluorescent aerosol PSL showed 99.9% ± 0.1 in the first channel and 0.1% in the
second channel, while 2.9% ± 0.1 of BF was found in the first channel and 96.5% ± 0.2 in
the last channel (the highest intensity channel [64]).
146
Table 4-2: Fluorescent percentage of fungal spores (Aspergillus and Penicillium) after seven days culturing time
(Aspergillus) (Penicillium) aDi.
Number of
fluorescent
spores (Mean)
Fluorescent %
Number of
fluorescent
spores (Mean)
Fluorescent %
1.84 11 58.9 ± 14.0
1.98 56 69.4 ± 6.1
2.13 159 77.2 ± 2.7
2.29 212 90.2 ± 2.6
2.46 179 97.2 ± 0.99
2.64 21 48.1 ± 13.7 174 99.0 ± 0.77
2.84 67 56.0 ± 12.9 102 99.1 ± 0.99
3.05 131 61.2 ± 11.0 48 97.8 ± 2.0
3.28 123 70.4 ± 4.3 16 90.2 ± 9.3
3.52 99 79.6 ± 5.4 8 76.1 ± 13.5
3.79 108 84.2 ± 8.0
4.07 70 88.4 ± 3.4
4.37 25 94.3 ± 4.6
4.70 9 93.3 ± 5.0 aAerodynamic diameter in µm.
However for Penicillium and Aspergillus species, fluorescent intensity distribution was
different, as expected, to the distribution for PSL and BF particles; but also different
between the two species. This can be seen from the results presented in Figure 4.5a for
Aspergillus and Penicillium species cultured for seven days, where Aspergillus fungal
spore percentage in the first four channels is higher than that for Penicillium spores. For
channels above the fourth, the inverse situation was obtained and in many of the channels
the Penicillium spore percentage was double that of Aspergillus.
147
However, when comparing spectra of the two species cultured for different periods of
times the situation changes. Figure 4.5b shows that the spectrum of seven-days-old
Aspergillus is almost identical to the spectrum of 14-day-old Penicillium. This indicates
the change of spectra with age of species and thus the importance of taking culture age
into account when comparing fungal species.
The second method of investigating spore fluorescent intensity utilized the spore
concentration percentage (for specific diameters in a single channel) versus channel
number. Spore concentration percentage, in this case, is the ratio of the spore
concentration of a specific diameter in one channel to the sum of spores of that specific
diameter in all 64 channels expressed in percentage. The results of application of this
method are presented in Figure 4.6 (including only every second value, to make the
diagram clear).
148
(a)
(b)
Figure 4.5: Spore concentration percentage (all diameters in single channel) as distributed between UVAPS channels: (a) Aspergillus and Penicillium species after seven days culturing time; (b) Aspergillus after seven days and Penicillium after 14 days culturing time.
The channel numbers on the UVAPS reflect the degree of fluorescence, with increasing
numbers reflecting an increase in the fluorescent intensity. It can be seen from Figures
4.6a and b that high concentrations were recorded for small spores in the lower intensity
channels (1-5), and low concentrations in the higher intensity channels (6-64); while the
inverse was found for the larger spores. The same trend was found between spore size and
their fluorescent intensity. Figure 4.6a illustrates, for example, that Aspergillus spores of
149
size 2.84 µm were present only up to channel 29, while the 3.79 and 4.37 µm spores were
present up to 51, and 64 channels, respectively. There was, with very limited exception, a
proportional relationship between the spore size and their concentration in the channels
from 6 to 64. On the other hand the results in channel 1, the non fluorescent channel,
showed an inverse relationship between spore concentration and its size, with 2.46 and
2.84 µm spores showing the highest concentration (of 50.3% and 44.3% respectively) and
4.37 µm spores, the lowest (at 5.8%).
For Penicillium, large spores (2.64 and 3.05 µm) can be found in all sixty-four channels,
with the spore concentration increasing in the higher channels with increasing fungal
spore size (Figure 4.6b). Spores of smaller diameters (1.98 and 2.29 µm), were found
mainly in the lower channels, with very low concentrations (less than 0.1%) present in the
higher intensity channels. For example spores with 1.98 µm diameters were present up to
channel 21, while the 2.29 µm diameter spores were present up to channels 40. In general,
from channel 15 to channel 64, the spore percentages were proportional to their sizes, i.e.
when the spore size increases, its concentration percentage in the higher channels
increase. The same trend between spore size and concentration as that observed for
Aspergillus was found in channel 1, with the exception of 3.05 µm diameter spores,
which showed a minor deviation from this relation.
It was observed that 21 -day-old fungal spores for both species were found at lower
fluorescent intensity channels than those that were seven-day-old (graphs not included).
The spore concentration decreased in the higher channels and increased in the lower
channels (1-5). For example, the concentration of the large Penicillium spores (3.05 µm)
in the last channel was 2.3% for seven days old spores, while it decreased to about 0.2%
for twenty-one-day-old spores.
150
(a)
0.01
0.1
1
10
100
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Channel number
Spo
re c
once
ntra
tion,
%
2.84 µm
3.28 µm
3.79 µm
4.37 µm
(b)
0.01
0.1
1
10
100
0 10 20 30 40 50 60
Channel number
Spo
re c
once
ntra
tion,
%
1.98 µm2.29 µm2.64 µm3.05 µm
Figure 4.6: Average spore concentration percentage (for specific diameter in a single channel) of seven day old spores as distributed between UVAPS channels (channel numbers reflect degree of fluorescence): (a) Aspergillus (b) Penicillium.
4.3.5 Fungal spore fluorescent percentage as a function of frequency of the exposure
The investigation of the effect of the frequency of the exposure to air on the fungal spore
fluorescent percentage showed that the percentage decreases with the increasing number
of exposure times (Figure 4.7). The section that was measured for the third time (exposed
three times to air current) showed the lowest fluorescent percentage (67.5% ± 5.4). The
section exposed twice showed a fluorescent percentage of 75.3 % ± 3.8, while the section
151
exposed only once (after fourteen days refrigeration) demonstrated the highest fluorescent
percentage (79.2% ± 3.9). Using the same procedure, Penicillium showed a similar
relationship (Figure 4.7).
50
60
70
80
90
1 2 3
Number of current air exposures
Fluo
resc
ent p
erce
ntag
e, %
AspergillusPenicillium
Figure 4.7: Fungal spore fluorescent percentage as a function to number of times exposed to air flow rate of 10 L/min.
4.4 Discussion
In this work fungal spore size and fluorescence of Aspergillus and Penicillium species
were investigated using the UVAPS. The spore sizes were studied as a function of fungal
spore age, while the fluorescence was measured as a function of age and number of air
exposures.
The concentration levels of the generated spores were not high enough to result in rapid
coagulation, which would interfere with the interpretation of the results of the study. The
ranges of the total spore concentrations, in all these studies except for two days culturing
time, were from 3.05 to 10.46 and from 3.56 to 14.4 particle/cm3 for Aspergillus and
Penicillium, respectively. The concentrations for two days culturing time were very small
(of the order of 0.2 #/cm3 after blank subtraction). A linear relationship between total
152
particle concentration and fluorescent particle concentration was identified for all of the
concentrations obtained in this study, as was shown in previous work (Kanaani et al.,
2006).
4.4.1 Fungal spore size as a function of age The change of spore size distribution for fungal species as a function of time is a
parameter that may help in the discrimination between the species. In this study, the mode
of the spore size distribution (Table 4.1) was investigated as a function of time under well
controlled conditions as a factor aiding in discrimination between Aspergillus and
Penicillium. However, analysis of the entire spore size distribution proved to be more
helpful in discriminating between the two species (Figure 4.3c).
While the location of the modes of Aspergillus and Penicillium spores of the same age,
i.e. 21-day-old (3.52 and 2.64 µm respectively), were quite different, the modes of
different ages, i.e. 4-day-old Aspergillus and 21-day old Penicillium spores, were less
different (3.05 and 2.64 µm). Thus, as the difference between their ages increased the
ability to discriminate between them decreased (Figure 4.3c).
4.4.2 The effect of age on fungal spore fluorescent percentage Figure 4.4 shows that the fluorescent percentage decreases for both fungal spores during
the culturing time, and that at all stages the fluorescent percentage of Penicillium is more
than that of Aspergillus. This may be interpreted as follows.
It has been reported that the viability of fungal spores decline from the moment that they
are released (Flannigan and Miller, 1994). Aspergillus and Penicillium spores have been
found to survive in dry air for decades (Flannigan and Miller, 1994), however in humid
environment, fungal spores’ viability declines with time. For example, Penicillium
chrysogenum spores subjected to moving air at 75% RH showed a reduction in
153
culturability (Muilenberg and Burge, 1994), and dehydrated N. crassa conidia have
remained viable for several years but they lost viability after nine days when stored under
conditions of 100% relative humidity (Griffin, 1994). In this work the relative humidity
during incubation, which was 60% ± 9, could have been the parameter resulting in the
decrease of fungal spore viability with age. The decrease in the levels of cofactors such as
nicotinamide-adenine dinucleotide (NADH), nicotinamide-adenine dinucleotide
phosphate (NADPH), and riboflavin with age is responsible for the decrease of
fluorescence signals.
The different fluorescent percentage between Aspergillus and Penicillium fungal spores
pointed out the possibility of using this parameter to differentiate between the two
species. However, when conducting the experiments under conditions similar to those
expected for a real home environment, and thus by varying culture age and air exposure,
the results were not so encouraging. Figure 4.4 shows that Aspergillus after four days had
the same fluorescent percentage as Penicillium after 14 days culturing time, and also that
14-day-old Aspergillus had the same fluorescent percentage as 21 days old Penicillium.
This indicates that it is not possible to differentiate between mixed populations of species
of mixed ages, as would be the case in a typical home environment.
For both Aspergillus and Penicillium, the increase of fluorescent percentage with the
increase in spore sizes, (Table 4.2), is likely to be a consequence of the increase of total
fluorescence amount with spore size. Hence the proportion of spores with fluorescence
exceeding the threshold detection baseline increased (as a results of spore growing), and
was detected by UVAPS as fluorescent particles, and subsequently located within 2-64
channels. This was the case for size channels with average or high number of spore
counts; but not for size channels with low spore counts (Table 4.2). This could be
154
explained as resulting from the influence of particle coincidence (phantom) (Heitbrink
and Baron, 1991; Holm et al., 1997) and the slower recovery of the UV laser compared to
the recovery of the APS laser of the UVAPS (Agranovski et al., 2003a), which leads to
lower estimates of the real fluorescent percentage. The effect of the last phenomenon was
obvious when the fluorescent particle number was low i.e. nine particles in the 4.70µm
and eight particles in the 3.52µm channels for Aspergillus and Penicillum, respectively
(Table 4.2).
4.4.3 The effect of age on the fungal spore fluorescent intensity All fungal spores contain the same sources of autofluorescence such as the reducing
fluorescent coenzymes nicotinamide-adenine dinucleotide (NADH) and nicotinamide-
adenine dinucleotide phosphate (NADPH), as well as the metabolic function riboflavin
(vitamin B2) (Li et al., 1991; Brosseau et al., 2000; Billinton and Knight, 2001).
However, fluorescent intensity of these biomolecules may vary according to the
environmental conditions under which the fungal colonies are placed, and due to their
concentration.
Under well-controlled conditions fluorescent intensity constitutes another parameter,
which helps to differentiate between fungal species under investigation. In order to
extract more information from spore fluorescence signals, spore fluorescence intensity
was considered according to two methods (Figures 4.5 and 4.6). The spectra, as shown in
Figure 4.5, aid in discriminating between the two species of the same age; but they are not
of much help for spores of mixed ages as would normally be the case in home
environment. In particular, for spores of the same age, spore size distributions (the
second method, see section 3.4.) were different for the two species with two major
differences, as can be concluded from inspection of Figure 4.6. Firstly, the spore
concentrations of Penicillium in higher intensity channels were more than that for
155
Aspergillus. Secondly, in contrast to Aspergillus, Penicillium was detected in all of the
higher intensity channels. The entire size distributions could serve as finger prints (or
signatures) for each of the species (Figure 4.6a and b). However, it was found that each
genus changed its fingerprint dramatically with age, which makes it difficult to
discriminate between mixed ages on the basis of their fluorescent intensity. In spite of
both spore species containing the same biomolecules (NAD(P)H)and riboflavin, they
were found to be distributed in different channels, which implies that the amount of these
biomolecules and their location within the spore are different for the same and different
species (i.e. same species but different age and different species of the same or different
ages). Under-well controlled laboratory condition the spore size distribution, together
with spore fluorescent percentage and intensity proved to be useful parameter in
differentiating between the species under investigation. However in ambient air, where
large populations of fungal species of different ages are present (Dix and Webster,
1995; Vijay et al., 1999b)as well as other biological and non biological airborne particles,
the task of differentiating between fungal spores, using the UVAPS, appears to be very
difficult, or even impossible.
4.4.4 The effect of air current on spore fluorescent percentage The results presented in Figure 4.7 demonstrate the effect of air current on fungal spore
fluorescence signals. The decrease in fluorescent percentage with the increase in the
number of exposures to air, for both of fungal spore types, was in agreement with a
previously reported study that investigated the effect of sampling time on fungal
culturability (Wang et al., 2001a). Wang et al.(2001a) showed that the relative
culturability of P. melinii and A. versicolor, using two personal filter samplers, was much
higher when sampled for 10 minutes than when sampled for 10 hours for both samplers.
Stanevich and Petersen (1990), also found that the viability of five-minute samples taken
156
with an Andersen N6 sampler was six times lower than that of one-minute samples. The
decrease in both culturability and fluorescent percentage is likely to be due to the impact
of exposure to air current and desiccation stress.
Fungal spores grow in different places in the indoor environment, and consequently are
subject to air exposure for different periods of time and under different air speeds.
According to the findings from this study, the variation in the number of exposure times
to the air, air speed and the duration of exposure periods will complicate the process of
differentiating between different fungal genera.
There are many other variables affecting fungal differentiation using the UVAPS such as
UV exposure, surfaces on which they are growing (for example timber, tile or gypsum)
and relative humidity, which will be the topics of future studies.
4.5 Conclusions
This study showed that the fungal spore size of the genera under investigation
(Aspergillus and Penicillium) increased with culturing time. Spore size distribution
helped as additional parameter in differentiating between the genera of the same age; but
not for genera of different ages. The fluorescent spore percentage decreased with
increasing fungal spore age and also with the number of times the fungal spores were
exposed to air currents. Based on fungal spore size distributions, together with fluorescent
percentages and intensities, the study demonstrated the ability of UVAPS to discriminate
between two fungal spore species under controlled laboratory conditions. In the field,
however, it would not be possible to use the UVAPS to differentiate between different
fungal spores due to the presence of different micro-organisms of varying ages and
subjected to different environmental conditions. In addition, the environment may contain
157
non biological aerosols which, when illuminated with the same wavelength as the spores,
will fluoresce, making the task of differentiation more difficult.
Acknowledgements
This project was supported by the grant number 1-9311-8174 from the Research Triangle
Institute, PO Box 12194 Research Triangle Park, NC 27709-2194. Special thanks go to
Dr David Ensor, Aerosol Science and Nanotechnology, whose vision made this study
possible. Support and assistance provided by ILAQH staff is gratefully acknowledged.
158
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CHAPTER 5 : Fungal spore fragmentation as a function of airflow rates and fungal generation methods
Hussein Kanaania, Megan Hargreavesb, Zoran Ristovskia and Lidia Morawskaa
aInternational Laboratory for Air Quality and Health, Queensland University of
Technology, Brisbane, QLD, Australia
bSchool of Life Sciences, Microbiology Section, Queensland University of Technology,
Brisbane, QLD, Australia and International Laboratory for Air Quality and Health
(2009) Atmospheric Environment 43: 3725-3735
164
Statement of Joint Authorship
Title: Fungal spore fragmentation as a function of airflow rates and fungal generation
methods
Authors: Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia Morawska
Hussein Kanaani: Developed experimental design; conducted experiments; analysed and
interpreted data and wrote the manuscript.
Megan Hargreaves: contributed to experimental design; interpreted data and offered
editorial comments during paper writing.
Zoran Ristovski: contributed to experimental design and scientific method; provided
technical assistance and helped in solving problems during the experiment.
Lidia Morawska: contributed to experimental design and helped in solving problems
during the experiment; interpreted data and offered editorial comments during paper
writing
165
Abstract
The aim of this study was to characterise and quantify the fungal fragment propagules
derived and released from several fungal species (Penicillium, Aspergillus niger and
Cladosporium cladosporioides) using different generation methods and different air
velocities over the colonies. Real-time fungal spore fragmentation was investigated using
an Ultraviolet Aerodynamic Particle Sizer (UVAPS) and a Scanning Mobility Particle
Sizer (SMPS). The study showed that there were significant differences (p < 0.01) in the
fragmentation percentage between different air velocities for the three generation
methods, namely the direct, the fan and the fungal spore source strength tester (FSSST)
methods. The percentage of fragmentation also proved to be dependant on fungal species.
The study found that there was no fragmentation for any of the fungal species at an air
velocity ≤ 0.4 m/s for any method of generation. Fluorescent signals, as well as
mathematical determination also showed that the fungal fragments were derived from
spores. Correlation analysis showed that the number of released fragments measured by
the UVAPS under controlled conditions can be predicted on the basis of the number of
spores, for Penicillium and Aspergillus niger, but not for Cladosporium cladosporioides.
The fluorescence percentage of fragment samples was found to be significantly different
to that of non-fragment samples (p < 0.0001) and the fragment sample fluorescence was
always less than that of the non-fragment samples. Size distribution and concentration of
fungal fragment particles were investigated qualitatively and quantitatively, by both
UVAPS and SMPS, and it was found that the UVAPS was more sensitive than the SMPS
for measuring small sample concentrations, and the results obtained from the UVAPS and
SMAS were not identical for the same samples.
Keywords: fungal spores; fragments; air velocity; fluorescent percentage
166
5.1 Introduction
Many published studies have confirmed the presence of indoor microbial contamination
around the world (Pei-Chih et al., 2000; Hargreaves et al., 2003c; Portnoy et al., 2004; Jo
and Seo, 2005). It has been reported that fungal spores are the cause of many adverse
health outcomes, such as asthma, rhinitis, allergies and hypersensitivity pneumonitis
(Vijay et al., 1999a), as well as infectious diseases such as dermatomycoses and
aspergillosis (Smith, 1976). It has also been found that submicrometer fungal fragments
have stronger adverse effects than fungal spores because they can penetrate deeper into
the respiratory tract and deposit into bronchi, bronchioles and alveoli (Cho et al., 2005).
Fungal spore release depends on several factors, including air velocity (air-flow rate over
the surface), relative humidity, temperature, building materials, fungal species,
ventilation, human activity and the age of mold growth. For example, Kildesø et al.
(2003) found that the relationship between released fungal spore numbers and air velocity
depends on fungal species, while Pasanen et al. (1991) found that spore release depends
on fungal genus and Górny et al. (2001) found that the release of fungal spores is affected
by fungal species, air velocity over the surface, surface texture and vibration of the
contaminated material.
In terms of fungal fragmentation, many studies have also investigated the potential
release and measurement of fungal propagules (Górny et al., 2001; Górny et al., 2003;
Górny, 2004; Cho et al., 2005). While Cho et al. (2005) investigated the aerodynamic
characteristics of the released fungal fragments, using a fungal spore source strength
tester, other studies investigated the effects of variables such as fungal species, airspeed,
167
humidity, vibration and colony structures, on the release of fungal fragments (Górny et
al., 2001; Górny et al., 2003; Górny, 2004).
Passive spore release, as a result of external forces such air currents and gravity is the
most common release mechanism for fungal spores in indoor environments (Gregory,
1973; Pasanen et al., 1991; Madelin and Madelin, 1995). Since air currents have been
identified as a major factor responsible for spore release (Burnett, 1976), it is expected
that indoor airflow velocities will also play an important role in this release, and
subsequently it may play a role in fungal fragmentation.
Airflow in indoor environments depends on the specific conditions of the environment.
For example, Handa and Pietrzyk (1996) showed that the airflow velocities for mixed or
displacement ventilation ranged from 0 to 0.3 m/s. Thorshauge (1982) found that the
mean air speeds in typically ventilated spaces, such as offices and lecture rooms, were in
the range 0.05-0.40 m/s, and Matthews et al. (1989) found the median air speed in four
typical residence houses was 0.06-0.16 m/s when the central fan was working and 0.02-
0.06 m/s when it was off. In order to account for outdoor air velocity (1.4-5.8 m/s (Górny
et al., 2001)), as well as mechanical and natural ventilation, the air velocities used in this
study ranged from 0.1-10.2 m/s.
While previous studies have investigated the effect of air velocity on fungal spore
aerosolization (Górny et al., 2003; Górny, 2004), they did not cover a wide range of air
velocities. The effect of different fungal releasing methods on fungal fragment release is
yet to be studied and it also remains unclear exactly which part of the fungus acts as the
source for fragment particles during the process of fungal fragmentation. As such, the aim
of this study was to characterise and quantify the released fungal fragment propagules
from different fungal strains (Penicillium, Aspergillus and Cladosporium), using different
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releasing methods and air velocities. The spore fragmentation mechanisms were also
investigated.
5.2 Materials and methods
5.2.1 Fungal preparation The fungal genera chosen (Aspergillus, Penicillium and Cladosporium) were those
frequently occurring indoors, in both Australia and other places in the world (Solomon,
1976; Kuo and Li, 1994; Burge et al., 2000; Pei-Chih et al., 2000; Hargreaves et al.,
2003c; Jo and Seo, 2005). Each of the Penicillium strain (Australian Collection of
Microorganisms - ACM 4616) and Aspergillus niger (American Type Culture Collection
- ATCC 9142) was inoculated onto Sabouraud Dextrose Agar plates (SDA), incubated at
25oC for two weeks and then refrigerated for one day before being used. Because the
culture obtained during the two week incubation period for Cladosporium
cladosporioides (Food Research Laboratory, CSIRO, Australia - FRR 5106) was not
sufficient to conduct the experiments properly, it was inoculated on SDA and incubated at
25oC for a further 3 weeks, and these samples were subsequently referred to as having a
“high fluorescent percentage” (see below) .
In order to investigate the difference in fluorescent percentage between fragmented and
non-fragmented samples, cultures with both “medium and low fluorescent percentages”
were also used for Aspergillus and Penicillium. As the fluorescent percentage of fungal
spores is known to decrease as they age, as well as with an increased frequency of air
exposure (Kanaani et al., 2007), these cultures were obtained as follows: after using the
high fluorescent percentage cultures for each species, the agar plates containing fungal
cultures were refrigerated for two months and then used again for generating fungal
aerosols with a medium fluorescent percentage. After using these medium fluorescent
169
percentage cultures, the agar plates were refrigerated for another two months and then
used again for generating fungal aerosols with low fluorescent percentage.
5.2.2 Instrumentation and applied protocol The tests were conducted at the International Laboratory for Air Quality and Health
(ILAQH) at Queensland University of Technology, inside a Class II, Type A, Biological
Safety Cabinet (SG-400 SterilGARD, E-mail Westinghouse Pty Ltd., Australia) over a
period of two years. Ten trials (each consisting of nine 3 hour experiments) were
conducted for each species of high percentage fluorescent level fungi, with one
experiment for each of the three methods of fungal particle generation. These trials used
the real-time Ultraviolet Aerodynamic Particle Sizer (UVAPS, model 3312, TSI Inc, St.
Paul., MN) for detecting and measuring fungal particles (spores and fragments), in order
to investigate the effects of using different releasing methods, along with different air
velocities, on the characterization, spore fragmentation mechanism and concentration of
the released fungal fragments from different fungal species. A further seven trials
(consisting of six 2.5 hour experiments, each) were also conducted for Penicillium and
Aspergillus with high percentage fluorescent level only at each of the three methods,
using the UVAPS, along with a TSI Model 3071A Scanning Mobility Particle Sizer
(SMPS) (TSI Inc., St. Paul, MN, USA) and a TSI Model 3025 Condensation Particle
Counter (CPC) (TSI Inc., St. Paul, MN, USA). These trials investigated full scale
fragment characterisation at two air velocities (0.4 m/s and 5.5 m/s), which were chosen
because 0.4 m/s represents the maximum air velocity of ventilated spaces (see
‘Introduction’) and 5.5 m/s was the highest common air velocity for the three methods.
Another seven trials (consisting of twenty one 1 hour experiments, each) were also
conducted to investigate the difference in fluorescent percentage between fragment and
non-fragment samples. The trials for Penicillium and Aspergillus were conducted for all
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percentage fluorescent levels, while for Cladosporium they were only conducted for the
high percentage fluorescent level. In these experiments, high, medium and low
fluorescent percentage cultures of each species were investigated using the UVAPS. All
of the trials were conducted using two air velocities (0.4 m/s and 5.5 m/s).
For each experiment, the background was measured using a petri dish containing only
SDA. In all cases, the SDA background showed a fluorescence of 0%, with very low
concentration levels (0 - 0.01 #/cm3), and as such, it was not necessary to control for the
background. The UVAPS sample time was 20 seconds for all of the experiments where
the UVAPS was used alone, and 120 seconds for the remaining trials, which also used an
SMPS, also with a sample time of 120 seconds. The UVAPS was calibrated using
standard particles, covering a range of 0.93 -7.17 µm aerodynamic diameter, as described
in Kanaani et al. (2007). The UV laser pulse energy and photomultiplier tube (PMT) were
kept constant during the study (50 ± 1% and 482 V respectively).
While the UVAPS can provide real-time concentrations, size distributions and
fluorescence for particles with aerodynamic diameters of 0.5-15 µm, the SMPS measures
particle size distribution and number concentration in the range 0.015-0.750 μm. Full
scale of airflow velocity measurements were carried out using a constant-temperature,
hot-wire anemometry air velocity meter (Model 8330-M-GB VelociCheck Air Velocity
Meter, TSI Inc., Cardigan Road, MN, USA), with an accuracy of ± 5% of reading or ±
0.025 m/s. The temperature and relative humidity range recorded during the study were
23-26oC and 65-69%, respectively.
5.2.3 Fungal aerosol generation methods Three dishes of each species were tested per day, one for each of the three methods of
generation (as outlined below).
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5.2.3.1 Generation of fungal aerosols by the direct method
The fungi were aerosolised directly from the cultures growing on the agar plates, which
were placed inside the mixing chamber (Figure 5.1a). During the experiments, continuous
HEPA-filtered air was directed over the surface of the mycelia at an angle of 60o and a
distance of 1.5 cm. Flow rates of 0.5, 2, 5, 9, 14, 20, 25 and 30 L/min (which correspond
to 0.1, 0.4, 1.8, 3.3, 5.3, 7.1, 8.5, and 10.2 m/s) were used. Sampling by the UVAPS
and/or UVAPS and SMPS (Figure 5.1) were conducted from the same sampling points
within the chamber during all experiments, in order to measure fungal propagule sizes,
concentration, fluorescent and total particle counts. The chamber used in the study was
100×39×39 cm and was made of aluminium, except for one side, which was made of
Perspex and used as a door.
5.2.3.2 Generation of fungal aerosols using the fan method
A small 12 volt fan, fixed on the top of one side of the chamber (from the inside), was
used to generate fungal particles (Figure 5.1b). The agar plate was held in place by a
stand and was directed towards the fan, so that they were both at the same level and that
the air hit the fungal colonies directly. The distance between the culture surface and the
fan was 2, 5, 15, 30 and 40 cm, in order to get the air blowing on the culture at different
speeds (0.1, 0.4, 1.8, 3.3 and 5.3 m/s, respectively).
5.2.3.3 Generation of fungal particles using the FSSST method
A small device, namely the fungal spore source strength tester (FSSST), was used to
release fungal particles (Figure 5.1c). FSSST is a portable device designed by
Sivasubramani et al. (2004b) and it is used to generate fungal particles by hitting their
colonies with clean air. The FSSST is a small four sided box, with an internal cross-
sectional area of 9.5 x 9.5 cm. It is made of polyvinyl chloride, with a 1 cm edge of foam
rubber around the open edge. It covers the petri dish and forms a seal with its rim, so that
172
no air can move in or out of the device, other than by the inlet and outlet. The clean,
HEPA-filtered air then enters the FSSST via a single opening, and exits via the 112
orifices, so that it is dispersed over the entire area of the fungal dish, which is located
directly under the device. The released fungal particles then leave the FSSST via the
outlet on its upper surface and are dispersed into the mixing chamber, to be collected and
analysed by the UVAPS and/or SMPS. The airflow rates used were 5, 10, 15, 25 and 30
L/min (corresponding to 0.1, 0.4, 1.8, 3.3, and 5.3 m/s, respectively), representing
different indoor environments (mechanical ventilation, domestic fan ventilation, natural
ventilation). For more information about the FSSST, see Grinshpun et al. (2002) and
Sivasubramani et al. (2004a).
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Figure 5.1: Experimental set-up for fungal aerosolisation: (a) Direct method; (b) Fan method; (c) Fungal spore source strength tester (FSSST) method, the remaining schematic drawing (biological safety cabinet, SMPS, UVAPS and HEPA filters) as in part (a). (d) Pressure equalizing holes.
(a)
( b )
( c )
HEPA Filter
Exhaust
Biological safety cabinet
HEPA Filter
Mixing chamber
Flow meter Petri dish _
_ _
(d) (d ) Air
SMPS UVAPS
HEPA Filter
Fungal growth
Fan
SMPS UVAPS
Biological safety cabinet
(d) (d )
Mixing chamber
Exhaust
FSSST upper side
Petri dish, under the orifices side of the FSSST
HEPA filtered air
Fungal particles out
(to be detected)
Mixing chamber
(d )
(d)
Fungal spore source strength tester (FSSST)
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5.2.4 Data analysis All statistical analysis, such as correlation and regression analysis were conducted using
Microsoft Office Excel 2003. Since the sample size (number of trials) was small (10 and
7), which is less than 15, the Mann-Whitney U test was run instead of the t-test. Because
of the potential effects of non-normality and a modest sample size on the ANOVA F
Statistic, the Kruskal-Wallis non-parameter test was also run to compare sample median
scores across the groups. The software package- SPSS for Windows (Version 16.0) was
used to conduct these tests. A level of significance p=0.05 was used for all statistical
procedures.
5.3 Results and Discussion
All figures and tables presented in this study represent the results of high fluorescent
percentage culture, except Table 3, which also includes the medium and low fluorescent
percentage results.
5.3.1 Related fungal fragment particles as a function of air velocity and generation methods
In all three generation methods (direct, fan and FSSST), fragmentation samples were
detected for all three forms of fungi under investigation (Penicillium, Aspergillus and
Cladosporium) (Figures 2). As shown in Figure 5.2, the fragmented fungal samples
consisted of particles smaller than those detected in non-fragmented samples for the same
species. This finding is consistent with the findings of previous studies that investigated
fungal spore fragmentation (Górny et al., 2002; Cho et al., 2005; Kanaani et al., 2008).
Table 1 presents the percentages of fungal fragmentation for different air velocities and
generation methods. The fragmentation percentage is defined as the number of samples
which contained fragmented spores as a percentage of the total number of samples at each
air velocity level. While fungal fragmentation began at airspeed of 1.8 m/s, for both the
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fan and FSSST methods, it started at 3.3 m/s for the direct method, for all of fungal
species under investigation (Table 1). The percentage of fungal fragmentation, using the
fan and FSSST methods, was much higher than using the direct method, for all air
velocity rates. For instance at an air velocity of 5.3 m/s, the percentage of fungal
fragmentation of Penicillium, Aspergillus and Cladosporium was 27.0, 10.5 and 14.5%
for fan method, 24.3, 10.4 and 11.2% for the FSSST method and only 0.5, 0.3 and 6.5%
for the direct method, respectively. This may be explained by the perpendicular angle of
the airflow to the culture, for both the fan and FSSST methods, which may have created
more turbulence and thus, a greater percentage of fragmentation and fungal spore release
(Górny et al., 2001).
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(a)
Figure 5.2: Typical UVAPS spectra for non-fragmented (Top) and fragmented fungal species (Below): (a) Penicillium species (ACM 4616).
177
(b)
Figure 5.2: Typical UVAPS spectra for non-fragmented (Top) and fragmented fungal species (Below): (b) Aspergillus niger (ATCC 9142).
178
(c)
Figure 5.2: Typical UVAPS spectra for non-fragmented (Top) and fragmented fungal species (Below): (c) Cladosporium cladosporoides (FRR 5106).
179
In general, the fragmentation percentage of the fungi increased with increasing air
velocity (Table 1). However, there were two exceptions using the direct method, one for
Penicillium (when the air velocity increased from 1.8 to 3.3 m/s) and the other for
Aspergillus (when the air velocity increased from 3.3 to 5.5 m/s) and one exception using
the fan method, for Penicillium (when the air velocity increased from 1.8 to 3.3 m/s). The
reasons behind these exceptions are unclear.
The Mann-Whitney U test demonstrated that there were significant differences (p < 0.01)
in the fragmentation percentage between 1.8, 3.3 and 5.3 m/s air velocities, for the three
generation methods. The Kruskal-Wallis non-parameter test also found significant
differences between the median scores (percentage of fragmentation) for each subsequent
increase in air velocity (according to species and generation method). As such, each test
was then followed by a Mann-Whitney U test and the differences remained significant (p
< 0.05), except in two cases for Penicillium, where the differences were not significant
(when the air velocity shifted from 1.8 to 3.3 m/s using the fan method (p = 0.97) and
when the air velocity shifted from 3.3 m/s to 5.3 m/s using the direct method (p = 0.14).
This increase in fragmentation percentage and number of released propagules, as a
function of air velocity, is consistent with the findings of previous studies (Górny et al.,
2003; Górny, 2004). Górny et al, (2003) reported a 14 fold increase in the release of
fragments in the actinomycete Streptomyces albus when increasing the air velocity on the
agar surface from 0.3 m/s to 29.1 m/s. Górny et al., (2002) also found a noticeable
increase in the number of released fragments for Aspergillus versicolor, Penicillium
melinii, and Cladosporium cladosporioides from the ceiling tile surfaces when air
velocity over colonies was shifted from 0.3 m/s to 29.1 m/s. However, this increase was
not observed when malt extract agar surfaces were used.
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Table 5-1: Percentage of fungal fragmentation as a function of air velocity and generation methods. __________________________________________________________________
Percentage of frequency fragmentation
Method Air velocity (m/s) Penicillium Aspergillus Cladosporium Fan 0.1 0.0 0.0 0.0
0.4 0.0 0.0 0.0
1.8 16.4 ± 5.3 5.5 ± 6.2 6.9 ± 4.2
3.3 15.1 ± 4.2 6.5 ± 4.7 8.1 ± 5.3
5.3 27.0 ± 7.6 10.5 ± 9.3 14.5 ± 7.9
FSSST 0.1 0.0 0.0 0.0
0.4 0.0 0.0 0.0
1.8 2.1 ± 2.0 0.9 ± 1.2 3.4 ± 2.1
3.3 6.8 ± 4.1 4.1 ± 3.9 6.0 ± 3.8
5.3 24.3 ± 9.3 10.4 ± 6.2 11.2 ± 6.1
Direct 0.1 0.0 0.0 0.0
0.4 0.0 0.0 0.0
1 .8 0.0 0.0 0.0
3.3 0.9 ± 0.9 0.8 ± 1.1 3.8 ± 2.1
5.3 0.5 ± 0.45 0.3 ± 0.5 6.5 ± 3.0
7.1 2.5 ± 2.3 0.7 ± 0.7 10.0 ± 4.8
8.5 7.6 ± 6.9 7.3 ± 5.3 12.7 ± 7.1
10.2 13.0 ± 10.2 11.1 ± 8.1 13.2 ± 6.9
__________________________________________________________________
Overall, the study found a significant correlation between fungal fragmentation
percentage and air velocity for all species and generation methods, except for Penicillium
(using the fan and FSSST methods) the correlation were not significant, where the p-
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value was slightly higher than 0.05: direct method: (r2 = 0.78, p < 0.05; r2 = 0.71, p < 0.05
and r2 = 0.94, p < 0.001 for the direct method for Penicillium, Aspergillus and
Cladosporium, respectively; r2 = 0.90, p < 0.05 and r2 = 0.92, p < 0.05 for the fan
method; and r2= 0.92, p < 0.05 and r2= 0.97, p < 0.005 for the FSSST method, for
Aspergillus and Cladosporium, respectively). The above results also demonstrated a more
significant association between fungal fragmentation and air velocity for Cladosporium,
compared to Penicillium and Aspergillus, for all generation methods. This may be
explained by the ellipsoidal to limoniform shape of Cladosporium spores (Samson and
Hoekstra, 1994), which fragments more easily than the globose shape of Penicillium and
Aspergillus spores (Raper et al., 1965; Ramirez, 1982). Table 1 also showed that the
fragmentation percentage of Cladosporium was more abundant than that of Penicillium
and Aspergillus, under the same conditions. The effect of the conidiophores shape (which
is smooth-walled and singlet or branched) in Penicillium and (singlet, smooth-walled) in
Aspergillus or (erect, straight unbranched or branched) in Cladosporium (Ellis et al.,
1992), together with effect of their colony surfaces on spore fragmentation need to be
investigated
All of the strains, for each generation method, showed zero fragmentation at 0.4 m/s,
indicating that for a typical indoor ventilation environment, where air velocity is ≤ 0.4
m/s (Thorshauge, 1982; Matthews et al., 1989; Handa and Pietrzyk, 1996), there would be
no fungal spore fragmentation for the species under investigation, except that which
occurs in the ventilation systems (ducts, pipes etc.), where the air speed can reach up to
29.1 m/s (Górny et al., 2002). While there was a low concentration of spores released at
0.1 and 0.4 m/s for Penicillium (around 0.7 and 1.7 #/cm3, respectively) and Aspergillus
(around 0.09 and 0.15 #/cm3, respectively), there were no spores released (0 #/cm3) for
Cladosporium. These results are consistent with a previous study by Pasanen et al.
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(1991), which reported that Cladosporium spores required a velocity of at least 1.0 m/s to
be released. However, Pasanen et al. (1991) also demonstrated that the release of A.
fumigatus and Pencillium sp. spores from conidiophores was initiated at an air velocity of
0.5 m/s, whereas this study found that they are released at a lower air velocity. This may
be due to the use of different Pencillium and Aspergillus species. In contrast, Górny et al.
(2002) found fungal fragments for Aspergillus versicolor, Penicillium melinii and
Cladosporium cladosporioides at 0.3 m/s. Again, this may due to the different species
(except Cladosporium cladosporioides) and agar which were used, as well as the different
sensitivities of the equipment used for measuring air velocity. It may also be due to the
lower relative humidity observed in Górny et al. (2002) (32-40%) (compared to 65-69%
in this study), since fungal propagules have been shown to be aerosolised more easily in
dry air (Pasanen et al., 1991; Foarde et al., 1993).
5.3.2 The characterisation and mechanism of fungal spore fragmentation In the previous study (Kanaani et al., 2008), the concentration of released fungal particles
was found to remain the same or decrease very slowly for successive samples of 20
seconds each. As such, any significant sudden change in concentration of particle number
was deemed to be the result of fragmentation. A typical example of fragmentation and
non-fragmentation for Penicillium is presented in Figure 5.3.
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1
10
100
1000
0 200 400 600 800 1000 1200Time, second
Tota
l par
ticle
con
cent
ratio
n, #
/cm
3 No Frag.
Direct
Fan
FSSST
Figure 5.3: The frequency of fragmentation of 60 successive samples (20 seconds each) for different aerosolisation methods for Penicillium species. (The ‘No Frag.’ data are presented for comparison purposes and are derived from using the fan method at 5.3 m/s).
As presented in Figure 5.3, 60 Penicillium samples were used to investigate the frequency
of fragmentation for the three generation methods, at the highest air velocity (direct
method at 10 m/s, fan and FSSST methods at 5.3 m/s). The highest air velocity for each
method was chosen as it gave the greatest percentage of fragmentation. The figure shows
that there is no apparent pattern to the occurrence of fungal fragmentation, with
frequencies ranging from ‘interval’ (many samples without fragmentation followed by
one fragmented sample) to ‘successive’ (repeating fragmented samples ranging from 2-5
occurrences) and even ‘no fragmentation for an extended period’ (no fragmentation in
more than 100 successive samples). Since temperature, humidity, air velocity, fungal
generation method and species were all constant, other factors such colony orientation or
the species strains and thickness of the conidiophores for each species may be responsible
for the frequency of fragmentation. Further investigations are necessary to determine the
reasons behind this phenomenon.
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Figure 5.4 shows Penicillium, Aspergillus and Cladosporium gave multimodal particle
size concentrations (3, 3 and 4 modes, respectively) when they fragmented, for each of
the three generation methods used. These modes were 0.54, 0.97 and 2.28 µm for
Penicillium, 0.54, 1.38 and 3.27 µm for Aspergillus and 0.54, 1.48, 2.28 and 3.27 µm for
Cladosporium, using the direct, fan and FSSST methods, respectively. It was also found
that the size of the modes of fragmented samples were associated with the mode size for
the non-fragmented samples (i.e. the larger the non-fragmented origin particles, the larger
the fragmented particles). For example, fragmented Aspergillus particles with a mode of
1.8 µm (1.4-2.6 µm) corresponded with non-fragmented origin particles with a mode of
3.3 µm (spore rang of 2.8-4.4 µm), while smaller fragmented particles with a mode of 1.3
µm (0.9-1.6 µm) corresponded with smaller non-fragmented original particles with a
mode of 2.8 µm (2.5-3.7 µm). In both cases, the size difference between the fragmented
and non-fragmented samples for each mode was 1.5 µm.
Figure 5.4 also shows that, in the case of both Penicillium and Aspergillus, the particle
size distribution modes were similar for each of the generation methods used for that
species, which suggests a specific fragmentation mechanism for each of these species.
Penicillium and Aspergillus are very closely related imperfect fungi of the same subclass
(Griffin, 1994) and both of them have round spores (Larone, 2002), the only major
difference being that Aspergillus spores are larger than Penicillium spores (which
therefore lead to larger modes after fragmentation, as described above) with longer
conidiophores and rougher colony surface (Ellis et al., 1992). Since larger fragmentation
modes were formed by Aspergillus than by Penicillium (Figure 5.4), this suggests that the
fungal particles which fragmented were in fact spores (more proves are listed, below, in
this section).
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Using the direct method of generation, at an air velocity of 3.3 m/s, the distribution was
bimodal for Penicillium and Aspergillus and trimodal for Cladosporium, with none of the
species displaying a submicrometer mode (i.e. 2.6 µm and 1.4 µm for Penicillium, 3.52
µm and 1.71 µm for Aspergillus and 3.5 µm, 1.6 µm and 1.3 µm for Cladosporium)
(Figure not shown). These results indicated that air velocity not only affects the
percentage of sample fragmentation, but it also affects the fragmentation mechanism.
An example of a typical individual sample for each species is shown in Table 2. As
shown in Table 2, the concentration of larger particles (spores) decreased after
fragmentation, while the concentration of smaller particles increased, for all of the species
investigated. This may due to the fact that the large spores have a larger surface area that
is exposed to the air current and therefore, they fragment more easily than the small
spores.
Table 2 also shows that after fragmentation, the large spores decreased in number and the
smaller ones increased (the small spores were mostly 0-4 before fragmentation). Directly
following fragmentation, the same mode and percentage of larger spores, as in the Post-
fragmentation sample, were also detected in the following non-fragmented sample. On
the other hand, the smaller particles which formed during fragmentation disappeared,
which means that the particles that underwent fragmentation were spores. While, our
results support the findings of Górny et al. (2002), who found that fragment samples of
Aspergillus and Penicillium species share common antigens with their spores, which
confirm the fungal origin of fragments, it also suggest spores as the source of fungal
propagules which produce these fragments.
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(a) Penicillium
0
10
20
30
40
0.4 4
parti
cle
conc
entra
tion,
%
particle diameter, µm
No Frag.DirectFanFSSST
(b) Aspergillus
0
4
8
12
16
0.4 4
Par
ticle
con
cent
ratio
n, %
Particle diameter, µm
No frag
Direct
Fan
FSSST
(c) Cladosporium
0
6
12
18
24
0.4 4
Par
ticle
con
cent
ratio
n, %
Particle diameter, µm
No Frag.DirectFanFSSST
Figure 5.4: Average particle concentration (for all 10 high fluorescent percentage experiments) of fragmented and non-fragmented fungal species for each of the three generation methods: (a) Penicillium; (b) Aspergillus; (c) Cladosporium.
187
Table 5-2: Typical samples of fungal spores under investigation; before and after fragmentation. Penicillium Aspergillus Cladosporium A. D.a N. F. S.b F.S.c N. F. S. F.S. N. F. S. F.S. 0.54 0 4 1 12 0 12 0.58 4 273 0 0 0 1 0.63 0 71 0 1 0 0 0.67 0 99 1 1 0 3 0.72 0 202 0 2 0 7 0.78 0 368 0 0 0 7 0.84 0 507 1 2 0 4 0.90 0 849 1 2 0 0 0.97 0 946 1 0 0 8 1.04 0 982 0 12 0 15 1.11 2 898 1 27 0 22 1.20 0 837 0 52 3 36 1.29 0 528 1 113 1 51 1.38 2 421 1 196 2 46 1.49 4 273 1 292 1 32 1.60 2 200 0 315 1 16 1.72 7 126 0 383 3 11 1.84 42 93 1 333 3 18 1.98 281 152 1 298 10 12 2.13 1146 359 2 207 14 20 2.29 2710 845 3 168 11 14 2.46 2480 867 3 75 13 9 2.64 1642 650 30 80 21 12 2.84 1058 534 141 138 64 52 3.05 404 333 483 402 159 134 3.28 131 183 747 551 165 143 3.52 56 93 688 463 131 76 3.79 19 51 562 393 82 52 4.07 12 20 606 411 39 33 4.37 17 30 481 336 21 15 4.70 14 18 223 154 8 5 5.05 11 22 60 45 6 6 5.43 11 21 20 21 0 0 5.83 9 19 5 5 0 1 6.26 11 15 4 6 2 3 6.73 4 24 4 6 0 2 7.23 4 20 6 7 3 4 7.77 3 9 4 7 2 8 8.35 6 8 3 5 0 2 8.98 3 5 9 7 0 1 9.65 4 13 1 4 0 1 10.37 3 4 1 5 1 0
aAerodynamic Diameter as measured by UVAPS. bNot fragmented sample. c Fragmented sample. (The large spore numbers for each species (shaded area) were decreased after fragmentation.)
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Fragmentation can lead to an increase in smaller particles by up to 5000 times (as found
in this study). The number of fragments depends on many factors, such as initial
concentration of the sample, air velocity, method of generation, fungal species and the
number of particles of interest before fragmentation (in this case it was the smaller
particles, which ranged between 0 - 4 particles per sample before fragmentation). For
example, a direct generation sample of Aspergillus, with an initial concentration of
4.4x102 #/cm3 and an initial particle count of 1.44x105 particles before fragmentation,
was found to have only 1 particle of 1.84 µm in diameter before fragmentation, whilst the
final concentration was found to be 5.6x102 #/cm3 and the final particle count was
1.84x105after fragmentation, with 4.67x103 particles of 1.84 µm in diameter. This
increase was among the highest found in this study.
Overall, the concentration and number of particles during this study increased after
fragmentation by a range of 1.01-1.92 times, depending on the conditions under which the
fragmentation occurred. For example, the mean increase in concentration and particle
number after fragmentation for all air velocities using the direct method, were 1.41 ± 0.27
and 1.39 ± 0.25 times for Penicillium, 1.34 ± 0.24 and 1.33 ± 0.25 times for Aspergillus,
and 1.21 ± 0.26 and 1.22 ± 0.24 times for Cladosporium.
Each of the large spores in an individual sample was found to have different fragment
spore percentages when compared to other spores in the same sample. The fragment spore
percentage is defined as the number of fragmented spores of a certain size as a percentage
of the non-fragmented spores of the same size in the original sample. To estimate the
spore fragment percentage in each sample, two assumptions were made: firstly, that the
spore fragment percentage of any spore size which increased in number after
fragmentation is negligible; and secondly, that any large spores of certain size found in
189
the fragmented sample represent the spores that did not fragment. For example, for
Penicillium (see Table 2), assumption one would meant that for 1.7 µm spores, an
increase from 7 to 126 fungal particles after fragmentation would give a spore fragment
percentage of 0.0 % (i.e. none of the original 7 spores underwent fragmentation), while
the second assumption would mean that for 1.98 µm spores, the decrease from 281 to 152
spores after fragmentation would give a spore fragment percentage of 45.9% (i.e. the 152
spores that remain after fragmentation are assumed to be unfragmented spores). Even
though assumption one may underestimate the spore fragment percentage and assumption
two may overestimate it, given that some or all of the post-fragmentation spores may
actually have originated from fragmented larger spores, Table 2 shows that in this study,
the first assumption is valid because most of smaller sizes had a particle concentration of
0.0 before fragmentation. Similarly for the second assumption, this was relevant only for
the larger spore sizes, which means that there would be very little contribution from
fragmented larger spores. The mean spore fragment percentage ranges of A. niger at
direct method at air velocities of 10.2 m/s, fan at 5.3 m/s and FSSST at 5.3 m/s were 23-
65%, 27-72% and 40-86%, respectively with high fragmentation percentage were found,
mostly, for mode spores and/or those close to the mode (i.e. one size above or below), for
all of the species investigated.
The fragmentation percentage of spores is defined as the number of fragmented spores as
a percentage of the total number of spores in the non-fragmented sample. The mean
fragmentation percentage of spores (mostly, the same diameters as those shaded spore
sizes in the examples shown in Table 2) at the highest air velocity applied in the study
(10.2 m/s for the direct method and 5.3 m/s for the fan and FSSST methods) were 61.6,
50.0 and 45.2% for the direct method, 53.0, 40.2 and 31.9% for the fan method, and 70.9,
53.3 and 53.4% for the FSSST method, for Penicillium Aspergillus and Cladosporium,
190
respectively. From these figures it can be seen that the spore fragmentation percentages
for the FSSST method were greater than for the other methods, for all of the species
investigated. This may be due to the fact that the colonies generated using the FSSST
method were under a small negative pressure (of a few Pascals), which may potentially
lead to a greater fragmentation percentage for those spores.
When only using the UVAPS, it was difficult to determine if each spore of the
investigated species was fragmented into three (or more) smaller parts, because if this was
so, submicrometer particles, total particle number and sample concentration would
dramatically increase, and this was not observed. Instead, it is hypothesised that each
spore fragmented into two larger parts, because while the particle number and sample
concentration increased in the range of 1.01-1.92, the total number of smaller particles did
not reach double the number of total fragmented spores. For instance, the mean number of
smaller particles which formed using direct method were 1.3 ± 0.31 times, 1.45 ± 0.26
times and 1.36 ± 0.42 times more than fragmented spores for Penicillium Aspergillus and
Cladosporium at 10.2 m/s, respectively. This suggests that the fragmented spores did not
divide into more than two large parts and that some of these spores fragmented so
completely that they could not be detected by the UVAPS (they were only detected by the
SMPS) (see Section 3.6.)
5.3.3 The expected impact of spore fragmentation on health
As shown in Figure 5.5, the largest portion of the fragmented sample for Penicillium
(46.4%) was in the range 0.6-0.99 µm, while for Aspergillus and Cladosporium the
largest portion of the sample (71.3% and 43.9%, respectively) was in the range 1-2 µm.
Due to fragmentation, fungal particles (spores) larger than 3 µm decreased significantly
after fragmentation for both Aspergillus and Cladosporium, while the submicrometer
particles (which have a greater impact on allergic conditions) increased 400 times, 9.4
191
times and 6.3 times for Penicillium, Aspergillus and Cladosporium, respectively. Since
Cho et al. (2005) showed that submicrometer fungal fragments are more likely than larger
fragments to be deposited in the alveolar region of the lung, especially in young children,
then it is expected the percentage deposition for Penicillium fragments in alveolar region
of the lung would be greater than that of Aspergillus and Cladosporium, under
comparable conditions. As fungal fragments have also been demonstrated to contain
mycotoxins (Brasel et al., 2005) and fungal antigens (Górny et al., 2002), this increase in
submicrometer fungal particles may be a significant contributor to the adverse health
effects associated with airborne fungal particles. As shown in Figure 5.5, spore
fragmentation also led to an increase in the percentage of fine particles (< 2.5 µm) and
these particles are more strongly associated with adverse health effects than coarse
particles (Dockery et al., 1993; Levy et al., 2000). For the above reasons, fungal
fragmentation needs to be taken into consideration when conducting future exposure
measurements.
192
1.0-2.0 µm62.1%
2.1-3.0 µm37.1%
<0.6 µm0.1%>3.0 µm
0.7%0.6-0.99 µm
0.1%
0.6-0.99 µm46.4%
1.0-2.0 µm30.2%
<0.6 µm15.5%
>3.0 µm1.4%
2.1-3.0 µm6.4%
(a)
0.6-0.99 µm0.2%
<0.6 µm0.0%
1.0-2.0 µm0.5%
2.1-3.0 µm67.0%
>3 µm32.3%
<0.6 µm1.4%>3 µm
7.2%
2.1-3.0 µm13.4%
0.6-0.99 µm6.6%
1.0-2.0 µm71.3%
(b)
1.0-2.0 µm3.0%
<0.6 µm0.1%
0.6-0.99 µm
0.5%
2.1-3.0 µm35.5%>3.0 µm
61.0%
2.1-3.0 µm30.9%
>3.0 µm21.4%
<0.6 µm1.3%
0.6-0.99 µm
2.5%
1.0-2.0 µm43.9%
(c)
Figure 5.5: The particle concentration percentage of fungal particles with (right side) and with out (left side) fragmentation as detected by UVAPS: (a) Penicillium; (b) Aspergillus; (c) Cladosporium.
193
5.3.4 The correlation between spores and fragment release Correlation analysis between the number of the spores and fragment release was
conducted for all species for each generation method at the highest air velocity (10.2 m/s
for the direct method and 5.3 m/s for fan and FSSST methods). The results demonstrated
a statistically significant correlation (for the direct and FSSST methods) between
Penicillium and Aspergillus fragments, and the spores detected by the UVAPS (r2 = 0.65,
0.30 and 0.52 for Penicillium and r2 = 0.77, 0.26 and 0.50 for Aspergillus, using the
direct, fan and FSSST methods, respectively (in all, cases: p < 0.05). However no
correlation was found between the spores and the fragments of Cladosporium, with the
correlation coefficients found to be very low for all of the generation methods (r2 = 0.13,
p = 0.47; r2 = 0.09, p = 0.65 and r2 = 0.15, p = 0.70 for the direct, fan and FSSST
methods, respectively). Previous studies have shown that the number of released
fragments can not be predicted on the basis of the number of spores released (Górny et
al., 2002; Górny et al., 2003), however, the methods used in these studies were not the
same for each set of experiments. Further, these studies classified the fragments for all
species as being between 0.3-1.6 µm, while in this study, fragmented sizes were
determined for each individual species and were found to range from 0.54-1.6 µm, 0.54-
1.98 µm and 0.54-1.84 µm for Penicillium, Aspergillus and Cladosporium, respectively.
In addition, the previous studies only looked at 30-min fungal propagule release (the time
of each experiment), while the correlation between spores and fragments in this study
were analysed for each sample (sample time 20 seconds).
5.3.5 Fungal particle fluorescent percentage In all species and for all methods, the fluorescent percentage of the fragmented samples
was lower than for the non-fragmented samples. For instance, using the fan method, the
fluorescent percentage of the fragmented samples was lower than that of the non-
194
fragmented samples by around 19.3%, 30.9% and 39.1% for Penicillium and 30.7%,
36.7% and 41.1% for Aspergillus, for high, medium and low fluorescent percentages,
respectively. The fluorescent percentage of fragmented samples was also 21.8% lower
than that of non-fragmented samples for Cladosporium, for high fluorescent percentages
(Table 3). Using the direct method, the decrease was similar to that of the fan method,
but the decrease was much less using the FSSST method (i.e. 9.1%, 13.7% and 12.3% for
Penicillium, Aspergillus and Cladosporium, for high fluorescent percentages,
respectively). Statistical analysis (Mann-Whitney U test) showed that there were
significant differences (p < 0.0001) between the fluorescent percentage of fragmented and
non-fragmented samples, for all of the species investigated, which is likely to be because
many of the fragmented particles were smaller than the threshold detection limit of the
UVAPS. On the other hand, the fluorescent percentage of fungal propagules decreased
with their decreasing size, for all species, and the submicrometer fragments were found to
have the lowest fluorescent percentage (Figure 5.6). These results agree with the previous
findings of Kanaani et al. (2007). It was also found that when the original fluorescent
percentage was smaller, the decrease in fluorescent percentage was actually greater (i.e.
the percentage decrease for low fluorescent species was greater than that of medium
fluorescent species, which was greater than that of high fluorescent species).
195
Table 5-3: Fluorescent percentage of fungal species with and with out fragmentation (fan method).
Fungal species Fluorescent percentage of
non fragment samples
Fluorescent percentage
of fragment samples
Penicillium 80.2 ± 1.7
50.1 ± 1.3
24.3 ± 1.4
65.0 ± 4.2
34.6 ± 4.8
14.8 ± 3.5
Aspergillus 58.2 ± 1.1
41.4 ± 0.9
23.6 ± 1.2
40.3 ± 5.8
26.2 ± 4.3
13.9 ± 2.0
Cladosporium 29.3 ± 1.5 22.9 ± 3.9
0
20
40
60
80
100
0.1 1 10
Particle diameter, um
Fluo
rese
nt p
erce
ntag
e, %
No fragDirectFanFSSST
Figure 5.6: Fluorescent percentages of Aspergillus (high fluorescent) particles as a function of particle diameters.
5.3.6 Comparison of size distribution of fungal fragmentation particles as measures by UVAPS and SMPS
Fungal fragmented particles were detected simultaneously by the UVAPS and SMPS
(Figure1). Most particle sizes (those less than 0.5 µm) that were beyond the detection
limits of UVAPS were detected by SMPS, so a full scale of fungal particles were
196
collected for both cases (fragmentation and not fragmentation). No signals were detected
by the SMPS for the background, non-fragmented Penicillium or Aspergillus species in
this study. Particle mobility diameter, dm, can be related to its aerodynamic diameter, da,
by introducing an effective density and it was corrected to aerodynamic diameter using
the following equation (Shi et al., 2001):
5.0
0
)()(⎟⎟⎠
⎞⎜⎜⎝
⎛=
mce
acam dC
dCdd
ρρ
where Cc(d) is the Cunningham slip correction, ρ 0 is unit density and ρ e is the effective
particle density. fungal spore density of 1.1 g/cm3 (Grinshpun et al., 2005) was used in
this equation for ρ e.
To compare similar particle sizes measured by the SMPS with that of the UVAPS, which
are located in the channels 0.542, 0.583, 0.626 and 0.673, the concentration of 533 and
552 nm particles detected in SMPS were summed together, since their mean was equal to
0.543 µm, which was comparable to channel 0.542 (µm) in the UVAPS. The same
calculations were conducted for the following pairs of particle sizes as measured by
SMPS: 573 and 594; 615 and 638; 661 and 685; and 710 and 737, in order to make
comparisons with channel sizes 0.583, 0.626 and 0.673 in the UVAPS.
Fragments of all sizes were detected by the SMPS (down to 0.02 µm) for Penicillium and
Aspergillus, however the number concentration percentage of ultrafine particle fragments
(less than 0.1 µm) for Penicillium and Aspergillus were very low (3.5 ± 3.6 and 4.7 ± 5.1
%, respectively) when compared to the rest of the range (0.1-0.737 µm) measured by
SMPS. Figure 5.7 shows a typical simultaneous sample for the SMPS and UVAPS. The
figure shows that the UVAPS was more sensitive than SMPS for detecting particles of
common diameters (0.54-0.723 μm). Generally speaking, it was found that when a signal
197
was detected by the SMPS, signal was also detected by the UVAPS, however the reverse
was not always the case. This means that the sensitivity of the UVAPS to detect particles
of certain diameter is greater than that of the SMPS, although, this sensitivity also
depends on the diameter of the particles being detected. For example, the minimum
concentration of 0.54 µm particles needed to be detected by the SMPS was greater than
that needed for larger particles (i.e. 0.58, 0.63 and 0.72 µm). Further, while 3.5 #/cm3 of
0.54 µm signal were detected by the UVAPS, no comparable signal was detected by the
SMPS, however many exceptions were reported in both cases. For example, the SMPS
was able to detect 0.1 #/cm3 of 0.54 µm particles, while concentrations as high as 0.74,
1.37, 2.2 and 1.94 #/cm3 for 0.58, 0.62, 0.67 and 0.723 µm particles detected by the
UVAPS produced no signals in the SMPS. On the other hand, concentrations of 0.06, 0.1,
0.15 and 0.2 #/cm3 for the same sized particles produced signals with the SMPS on
numerous occasions. In order to determine the reasons behind these behaviours, and to
quantify the total fragments detected in both SMPS and UVAPS, more detailed
investigations need to be conducted in the future.
198
0
100
200
300
400
500
600
700
800
0 0.2 0.4 0.6 0.8Aerodynamic diameter, µm
dN/d
logD
p (#
/cm
3 ), S
MP
S
0
10
20
30
40
50
60
70
80
90
dN/d
logD
p (#
/cm
3 ), U
VA
PS
PeAsPe UVAPSAs UVAPS
Figure 5.7: Typical samples of Penicillium and Aspergillus detected and measured by SMPS and UVAPS, simultaneously.
5.4 Conclusions
This study investigated the effects of using three different generation methods (direct, fan
and FSSST), with different air velocity levels (0.1-10.2 m/s), on the characterisation,
spore fragmentation mechanism and concentration of the released fungal fragments for
Aspergillus, Penicillium and Cladosporium, using a UVAPS. While most of the air
velocity levels used in this study produced detectable fungal particles for all the three
species and generation methods, no particle fragments were detected at 0.1 and 0.4m/s
(i.e. the air velocities which represent normal indoor ventilation environments). The study
showed a significant correlation between fungal fragmentation percentage and air
velocity, (r2 = 0.78, p < 0.05; r2 = 0.71, p < 0.05 and r2 = 0.94, p < 0.001 for the direct
method for Penicillium, Aspergillus and Cladosporium, respectively; r2 = 0.90, p < 0.05
and r2 = 0.92, p < 0.05 for the fan method; and r2= 0.92, p < 0.05 and r2= 0.97, p <
0.005 for the FSSST method, for Aspergillus and Cladosporium, respectively). The
specific parts of the fungal colony which were found to undergo fragmentation were the
SMPS
SMPS
199
fungal spores. The results of this study also suggest that these fungal spores either
fragmented into two larger particles, along with many smaller submicrometer particles or
they fragmented into submicrometer particles only. Using the UVAPS, the submicrometer
particles (which have a greater impact on health) were found to increase by up to 400
times, 9.4 times and 6.3 times for Penicillium, Aspergillus and Cladosporium,
respectively, during fragmentation. It was also found that, for all species and methods, the
fluorescent percentage of the fragmented samples was lower than that of the non-
fragmented samples. On the other hand, the full scale of fungal particles produced for
both fragmented and non-fragmented samples were detected, using a combination of
UVAPS and SMPS, and the concentration of ultrafine particle fragments for Penicillium
and Aspergillus were found to be very low when compared to the rest of the range (0.1-
0.737 µm) measured by the SMPS.
Acknowledgements
This project was supported by Grant 1-9311-8174 from the Research Triangle Institute,
Research Triangle Park, NC 27709-2194. Special thanks go to Dr David Ensor, Aerosol
Science and Nanotechnology, whose vision made this study possible. The financial
support Queensland University of Technology for one year is highly appreciated. The
support provided by ILAQH staff and the assistance of senior research support specialist,
Mr Ray Duplock, in relation to the statistical analysis performed in the study are also
gratefully acknowledged.
200
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CHAPTER 6 : Deposition rates of fungal spores in indoor environments, factors effecting them and
comparison with nonbiological aerosols.
Hussein Kanaania, Megan Hargreavesa, b, Zoran Ristovskia
and Lidia Morawskaa
aInternational Laboratory for Air Quality and Health, Queensland University of
Technology, Brisbane, QLD, Australia
bSchool of Life Sciences, Microbiology Section, Queensland University of
Technology, Brisbane, QLD, Australia
(2008) Atmospheric Environment 42: 7141-7154
206
Statement of Joint Authorship
Title: Deposition Rates Of Fungal Spores In Indoor Environments, Factors Effecting
Them And Comparison With Non-Biological Aerosols.
Authors: Hussein Kanaani, Megan Hargreaves, Zoran Ristovski and Lidia Morawska
Hussein Kanaani: Developed experimental design and scientific method; conducted
experiments; analysed and interpreted data; wrote the manuscript.
Megan Hargreaves: contributed to experimental design, interpreted data and offered
editorial comments during paper writing.
Zoran Ristovski: contributed to experimental design and interpreted data; provided
technical assistance and contributed in solving problems during the experiment.
Lidia Morawska: contributed to experimental design and helped in solving problems
during the experiment; interpreted data and offered editorial comments during
manuscript writing.
207
Abstract
Particle deposition indoors is one of the most important factors that determine the
effect of particle exposure on human health. While many studies have investigated the
particle deposition of non-biological aerosols, few have investigated biological
aerosols and even fewer have studied fungal spore deposition indoors. The purpose of
this study was, for the first time, to investigate the deposition rates of fungal particles
in a chamber of 20.4 m3 simulating indoor environments by: 1) releasing fungal
particles into the chamber, in sufficient concentrations so the particle deposition rates
can be statistically analysed; 2) comparing the obtained deposition rates with non-
bioaerosol particles of similar sizes, investigated under the same conditions; and 3)
investigating the effects of ventilation on the particle deposition rates. The study was
conducted for a wide size range of particle sizes (0.54 – 6.24 µm), at three different
air exchange rates (0.009, 1.75 and 2.5 h-1). An Ultraviolet Aerodynamic Particle
Sizer Spectrometer (UVAPS) was used to monitor the particle concentration decay
rate. The study showed that the deposition rates of fungal spores (Aspergillus niger
and Penicillium species) and the other aerosols (canola oil and talcum powder) were
similar, especially at very low air exchange rates (in the order of 0.009). Both the
aerosol and the bioaerosol deposition rates were found to be a function of particle
size. The results also showed increasing deposition rates with increasing ventilation
rates, for all particles under investigation. These conclusions are important in
understanding the dynamics of fungal spores in the air.
Keywords: deposition rate; air exchange rates (AER); fungal particles; fluorescent percentage; ventilation rate
208
6.1 Introduction
Indoor fungal spore exposure has been associated with adverse respiratory and
allergic health (Cuijpers et al., 1995; Verhoeff and Burge, 1997b). Since many people
spend most of their time indoors, in locations such as homes, offices and factories
(Ott, 1982; Lebowitz, 1983; Byrne, 1998; Brasche and Bischof, 2005), concern
regarding the health effects of indoor air quality has grown. Yet, little is known about
the dynamics and the role of biological aerosols in indoor environments, including
fungal spores, where dynamics include such processes as particle collision,
coagulation and deposition.
Deposition of particles on indoor surfaces is one of the most important elements of
particle dynamics (Lai, 2002) and ventilation is one of the key factors to affect
particle deposition rates indoors (Jamriska et al., 2000; Howard-Reed et al., 2003;
Wallace et al., 2004a). While a large number of studies have been conducted to
determine the deposition rates of non-biological aerosols in indoor environments
(Byrne et al., 1995; Thatcher and Layton, 1995; Fogh et al., 1997; Long et al., 2001;
Mosley et al., 2001; Vette et al., 2001; Thatcher et al., 2002; Howard-Reed et al.,
2003; Ferro et al., 2004; He et al., 2004; Wallace et al., 2004a; He et al., 2005), very
little work has been done with regard to the deposition rate of fungal spores (Reck et
al., 2002). One exception was a study by Reck et al. (2002), who investigated,
experimentally and numerically, the deposition of spoilage fungi in a petri dish, on a
surface and on a warm box-shaped object placed in a food-processing environment.
However, as yet, the deposition rates of fungal spores in a room-sized chamber have
not been investigated.
209
On the other hand, several studies have investigated the effect of ventilation on
particle concentrations indoors (Jamriska et al., 2000; Howard-Reed et al., 2003;
Wallace et al., 2004a), while Jamriska et al. (2000) investigated the effect of
ventilation on the reduction of submicrometer particle concentration in occupied
office building. Howard-Reed et al. (2003) and Wallace et al. (2004a) quantified the
particle loss rate in occupied townhouses due to the operation of central fans.
However, none of them examined the effect of ventilation on particle deposition rates
for bioaerosols.
Therefore, the aims of this study were to: 1) study the deposition rates of Aspergillus
niger and Penicillium spores in a chamber with no ventilation; 2) study this deposition
rate for two different air exchange rates (AER) using a mechanical ventilation system;
and 3) compare the obtained results with the results of non-bioaerosol particles
(canola oil and talcum powder) examined under the same conditions. The real-time
UVAPS instrument was used in this study, which is its first reported application to
study the dynamics of bioaerosols.
6.2 Material and Methods
6.2.1 Experimental chamber and mechanical ventilation system This work was performed in an experimental chamber designed for the purpose of this
study, located at the Queensland University of Technology, in Brisbane, Australia.
The chamber, made of plywood and painted with water proof paint, had dimensions of
2.38×3.57×2.40 m, with a volume of 20.4 m3 and a surface to volume ratio of 2.23 m-
1. It had one door and three windows (Figure 6.1), to simulate a real indoor
environment and the chamber size and the ventilation system designed for it were
similar to a small indoor room or office. A mechanical ventilation system (MVS)
210
(Type: HRU SV-L 009, Poland), with the heat exchange disabled, was connected to
the chamber and the ambient supply air entered the ventilation system through a large
high efficiency HEPA filter (Figure 6.1). The air was sucked by a fan and transported
by both steel and flexible aluminium ducts, to enter the chamber through one louver
face diffuser, with three slots, to distribute the air in all directions. The air then left the
chamber through a return-air grill in its ceiling and was transported through the steel
and flexible ducts, to enter a large high efficiency HEPA filter. The HEPA filtered air
then entered the MVS (all with zero fraction return) and was returned to the
atmosphere via a second fan, such that the MVS worked as mixed supply/exhaust
system. This mixed ventilation system, with louver faced diffusers and return air
grills, was chosen because such systems are commonly used worldwide.
During all measurements, the background particle concentration (inside the chamber)
was in the range of 0.26-0.57 #/cm3 before starting the experiments and the chamber
was well sealed, with the windows and door jambs, cracks and holes sealed with
plastic tape.
211
Figure 6.1: Experimental set-up.
6.2.2 Air speed, air flow pattern, temperature and humidity monitoring
Full scale measurements of air flow velocities were carried out in the chamber with
and without ventilation, using air velocity meter (Model 8330-M-GB VelociCheck
Fungal particle out from one of the five opening
Petri dish with fungal colonies (shown empty)
Air pumps
_
Exhaust
Ventilation system
Air HEPA filtered
air into
UV-
Fungal spore source strength testers
(FSSST), (The side with the orifice)
Table
Four Petri dishes under
_
_
Air in
Experimental chamber
Flow Window
DoorWindow
Window
HEPA
Petri dish with fungal
Small
HEPA filtered air
212
Air Velocity Meter, TSI Inc., Cardigan Road, MN, USA). The instrument is a
constant-temperature hot-wire anemometer and its accuracy is ±5% of the reading, or
± 0.025 m/s. The measurements were conducted on a 4 x 4 x 3 grid (length x width x
height), where the heights were 0.6, 1.1 and 1.7 m from the floor, as recommended by
the ISO standard 7726 (Hanzawa et al., 1987). The chamber volume was divided in a
grid-like manner, into 48 cubes and the measurement locations were in the centre of
each of these cubes. The air flow pattern was assessed by releasing smoke into the
space, using smoke candles and a Dyna-Fog Cyclone (Model No. 2732, Curtis Dyna-
Fog Ltd., Westfield, Indiana, USA) (Persily, 2005). The pressure difference between
the inside and the outside of the chamber was measured using the Differential
Pressure Meter Micromanometer (Model 8705 DP-CALCTM, TSI Incorporated, MN,
USA), with an accuracy of 1% of the reading, ± 1Pa.
6.2.3 CO2 measurements and air exchange rate (AER) estimation When used as tracers, both CO2 and SF6 have shown a good correlation with each
other (Ekberg and Strindehag, 1996; Mai et al., 2003; He et al., 2004) and since the
CO2 method is cheaper and more environmentally friendly, it was chosen for the AER
measurements in this study. The AER measurements were performed under three
different conditions: 1) with the ventilation system switched off (V0), 2) with the air
ventilation system switched on (V1); and 3) with the air ventilation system operating
at higher ventilation rate (V2). The different ventilation rates were obtained by
changing the voltage of MVS speed controller. Hereafter, airflow rates of V1 and V2 is
represented by ‘AER V1 and AER V2’, respectively. The CO2 tracer gas method was
performed by injecting the CO2 gas and mixing it through the chamber, using the
mechanical ventilation system. For AER measurements at V0, the MVS was switched
on for 1-2 minutes, to allow for the mixing of the gas, before it was switched off. For
213
V1 and V2, the system remained on after the initial mixing was conducted as for V0.
The CO2 concentration decay rate was measured by the TSI Model 8551 Q-Trak
instrument (TSI Incorporated, St. Paul, MN, USA). The CO2 concentration peak in
these experiments was around 1500 ppm, which decreased uniformly until it reached
the background concentration (415-430 ppm). CO2 concentrations were measured for
periods of around 120 h, 30 min and 20 min, with a time resolution of 1 minute for V0,
V1, and V2, respectively.
Under uniform mixing conditions, in the absence of chemical reactions between the
gas and other chemicals and in the absence of indoor sources, the air exchange rate (α)
can be estimated as follows (Nantka, 1990):
0
ln1CC
tt=α (1)
where t is the time in hours; Ct and C0 are the concentration of CO2 gas at time t and 0,
respectively. Based on Eq. (1), the AER value was obtained by linear regression of the
decay rate of the CO2 concentration for each experiment. The AER estimation was
conducted six times for each set of ventilation conditions.
6.2.4 The UVAPS instrument The UVAPS provides particle count size distributions, as well as real-time
fluorescence for particles with aerodynamic diameters of 0.5–15 µm. Fluorescence
measurements are produced by exciting particles with an UV laser beam at a
wavelength of 355 nm and then detecting the fluorescence emission from 420 to 575
nm. Using the UVAPS, aerosols with no fluorescent compounds appear in channel 1,
with a very small fraction appearing in channel 2, while the bioaerosols (with
endogenous metabolites, and thus fluorescence) appear in channels 2 to 64.
214
The UVAPS was calibrated using the 0.993 µm diameter monodispersed Polystyrene
Latex Particles and 0.91 µm diameter Blue Fluorescent microspheres (Duke Scientific
Corporation, Palo Alto, CA, USA). Both were aerosolised using the Collison
nebuliser - for details see (Kanaani et al., 2008). A monosized polymer particle
suspension, with 0.1% solid standards (SS-2-PXG 0.1%), (SS-5-PXG 0.1%) and (SS-
7-PXG 0.1%), and 1.05 g/cm3 of Dyno Particles, was also used according to the
factory calibration procedure (dry redispersion). Since the fluorescence spectra
detected by the UVAPS are very sensitive to the changes in the UV laser pulse energy
and photomultiplier tube (PMT) gain (TSI-Incorporated, 2000), the detected threshold
base line was controlled and checked during the course of the experiments. The UV
laser pulse energy and the PMT gain were set to 50 ± 1% of the laser power and 500
V, respectively. The UVAPS is the only instrument that measures the emitted
fluorescence signals of bioaerosol particles in real-time. It is also the most suitable for
indoor studies because it has a low flow rate (so it has negligible impact on particle
concentrations), quiet operation and short-sampling times (20 s).
6.2.5 Sample preparation Aspergillus niger (American Type Culture Collection - ATCC 9142) and Penicillium
sp. (Australian Collection of Microorganisms - ACM 4616) were inoculated on
Sabouraud Dextrose Agar (SDA) and incubated at 25oC for four and two weeks,
respectively, then refrigerated for one day before being used for aerosolization by a
direct generation method. Hereafter, Aspergillus niger (ATCC 9142) and Penicillium
sp. (ACM 4616) are referred to as Aspergillus and Penicillium, respectively. These
two fungi represent the bioaerosol samples, while the non-bioaerosol samples were
canola oil (Home Brand Non-Stick Canola Oil Spray) and talcum powder (Fleurique
Talcum Powder, made in Victoria, Australia). Both of the non bioaerosol samples
215
were purchased from Woolworths Limited Company, Australia. These aerosols were
chosen to cover a wide size range (0.54-6.26 µm) of particles in the inhalable range
(Li and Kuo, 1994; DeKoster and Thorne, 1995).
6.2.6 Aerosol generation and measurement Before the commencement of particle generation, the MVS was operated at V1 for a
period of 1-1.5 hours, supplying clean HEPA filtered air to the chamber, in order to
bring the background particle concentration to 0.26-0.57 #/cm3. After the background
particle concentration reached the desired level, particle generation was started. A
specific effort was made to release the fungal spores in sufficient concentration, so
that the fungal particles were more than 10 times the background particle
concentration for each experiment. To release Aspergillus particles in the chamber,
the following protocol was applied: six square large Petri dishes of 100mm x100mm x
20mm (length x width x height), each containing confluent fungal colonies, were
placed on a solid horizontal surface (table) before the required ventilation conditions
were initiated (see Figure 6.1).
Two concurrent methods were used to release the particles. Firstly, a narrow jet of
HEPA filtered air was aimed at the surface of the Aspergillus mycelia in one of the six
dishes, at an angle of 60o and a distance of 1.5 cm - for more details, see Kanaani et
al. (2007). Secondly, two fungal spore source strength testers (FSSST) were used to
release fungal particles from the five remaining Petri dishes. FSSST is a portable
devise designed by Sivasubramani et al. (2004b) and used to generate fungal particles
by exposing their colonies to clean air. The FSSST was made of polyvinyl chloride,
with a 1 cm edge of foam rubber around it, which completely covered the Petri dishes
and sealed them so that there was no air movement, other than from the inlet and the
outlets. The air outlet on one of the FSSST used was enlarged, in order to release
216
fungal particles from four Petri dishes at the same time, while the remaining FSSST
was used to release fungal particles from the remaining dish on its own. The clean,
HEPA-filtered air that entered the FSSST through a single inlet was distributed over
the internal cross-sectional area and directed towards fungal spore colonies through its
orifices (Figure 6.1). The large FSSST had an internal cross-sectional area of 30 x 30
cm with 572 orifices, whilst the small FSSST had an internal cross-sectional area of
9.5 x 9.5 cm with 112 orifices.
The fungal particles were released to the chamber from the FSSST outlets (see Figure
6.1 and for more details see Górny et al. (2001)). Three separate pumps were used to
supply the air through flexible tubes to the HEPA filters, which then passed through
the flow meter, before hitting the surface of the colonies, either directly, or through
the FSSST devices. The pumps were operated with flow rates of 20, 27 and 50 L/min,
for direct method, small FSSST and large FSSST, respectively. The increase of
particle concentration was monitored by the UVAPS and the pumps were turned off
when the concentration peak was reached. The generation time for the Aspergillus
colonies was 3 minutes in the first run and 15 minutes in the second run, which was
expected because fungal colonies used for the first time needed much less time to
reach the peak concentrations than colonies used for a second time. The same protocol
was followed for Penicillium, except that the generation times were lower (2 and 10
minutes for the first and second runs, respectively), since the colony was more dense
than that of Aspergillus. The MVS was either turned off (V0), left running as it was
(V1) or turned up (V2), depending on which experiment was being conducted. The
UVAPS measurements continued until the particle concentration returned to
background levels.
217
Canola oil particles were generated by shaking the can well and spraying the oil inside
the chamber through a hole in the wall, in the same location that the fungal particles
were aerosolized. Two to three sprays were used to reach the peak concentration and
the same ventilation conditions were used as for the fungal particle generation.
For talcum powder, the same steps were followed as per the fungal particle
generation, except that the powder, which was placed in one empty Petri dish, was
released by directing a narrow jet of HEPA air onto the surface of the powder at an
angle of 60o and a distance of 1.5 cm. A pump was used to supply the air, with flow
rate of 50 L/min, over a period of 0.5-1 minute.
Care was taken so that the generation location, pump flow rates and the angle of air
flow were as similar as possible for each experiment, so that the initial speed and
direction of particles generated were the same for all experiments. Each aerosol or
bioaerosol experiment was conducted seven times for each set of ventilation
conditions. All statistical analysis such as correlation and regression were conducted
using Microsoft Office Excel 2003. Since sample size (number of trials) was small
(7), Mann-Whitney U test was run in stead of t-test using software package- SPSS for
Windows (Version 16.0). A level of significance p=0.05 was used for all statistical
procedures. Nobody was inside the chamber during the course of the experiments,
which were monitored by the UVAPS and observed from outside through the
chamber window.
6.2.7 Fungal particle identification
Microscope slides covered with pieces of transparent adhesive tape (sticky side up)
were placed inside the chamber, in order to capture the released particles. The
released fungal particles were then observed under an optical light microscope (Model
218
CX31RTSF, Olympus Corporation, Tokyo, Japan) to identify whether they were
spores or not.
6.2.8 Estimation of particle deposition rates
6.2.8.1 Ventilation system off
For particle sizes similar to those investigated in this study (0.54-6.26µm), the
deposition rate on indoor surfaces was determined mainly by the gravitational settling
velocity. Assuming well-mixed conditions, the rate of change in indoor concentration
of any particle size with respect to time can be expressed using the following mass
balance equation (Koutrakis et al., 1992; Chen et al., 2000; Thatcher et al., 2002;
Wallace et al., 2004b; He et al., 2005):
dpidpdpivdpodpdpi CCGCf
dtdC
,,,, βαα −−+= (2)
where α is the air exchange rate (h-1); fdp is the penetration efficiency of the interest
particle diameter (dimensionless); Co is the outdoor concentration (outside the
chamber) at time t (particles/cm3); G is the generation of particles indoors
(particles/cm3 h); Ci is the indoor concentration at time t (particles/cm3); β is the
deposition rate coefficient (h-1); and dp is diameter of the particle of interest
(micrometers). Since the particle concentration was low, the coagulation was ignored
and not included in Eq. (2). Chemical reaction and hygroscopic growth were also
ignored, as they were not relevant to the controlled experimental conditions of the
study.
In the absence of the indoor particle sources (after reaching the peak concentration,
the particle generation was seized), Eq. (2) can be written as follows:
219
dpidpdpidpodpdpi CCCf
dtdC
,,,, βαα −−= (3)
Since the experiment was conducted in a tightly sealed chamber, the air exchange rate
(α) was very small and could be ignored. In addition, the particle concentrations
during aerosol generation were significantly higher than the background level, thus
the contribution from outdoor sources was very small and could also be ignored.
Assuming that α and βdp are constants, the time dependent solution to Eq. (2)
becomes:
tCC
dpdpi
dpi )()ln()0(,
, βα +−= (4)
where the Ci,dp(0) is the peak indoor particle concentration, i.e., t =0. Based on Eq. (4),
βdp can be determined by fitting a line to the plot of the natural log of Ci,dp/Ci,dp(0)
versus time and subtracting α from the slope.
6.2.8.2 Ventilation system on
Assuming well-mixed ventilated conditions, the infiltration from outdoors, other than
from ventilation (α), was negligible when compared to ventilation rate (175 times
lower) and when no particle generation or coagulation is occurring in the chamber
during the decay, the rate of change in indoor concentration of any particle size, with
respect to time, can be written as follows:
dpidpdpivdpovdpi CCC
dtdC
,,,, βλλ −−= (5)
where, λv is ventilation air exchange rate (h-1). Since the ventilation system was
equipped with a HEPA filter, the particle infiltration from outdoors can be ignored.
220
Assuming that λv and βdp are constants, the time dependent solution to Eq. (5)
becomes:
(6)
And again, βdp can be determined by fitting a line to plot of the natural log of
Ci,dp/Ci,dp(0) versus time and subtracting λv from the slope
6.3 Results
6.3.1 Air exchange rates, airspeed and airflow pattern The AER measurements in the experimental chamber were estimated using a CO2 gas
tracer. The obtained average results were 0.009 ± 0.003, 1.75 ± 0.32 and 2.50 ± 0.18
h-1 for V0, V1 and V2, respectively.
The mean air speeds estimated in this study were 0.01±0 .01, 0.15 ± 0.10 and 0.30
±0.12 m/s for V0, V1, and V2, respectively. The standard deviation represents the
variation in the flow among all the 48 points in the grid for each ventilation rate. The
mean air speed represents the air speed at the centre of the chamber, equidistant from
the walls, floor and ceiling.
At V0, the air speed at all 48 measurement sites ranged from 0.0-0.02 m/s. Maximum
airspeeds were found to be concentrated close to the inlet diffuser. For V1 and V2, the
results ranged from 0.02 to 0.32 m/s and 0.30 ±0.12 m/s, respectively, with higher
airspeed in the centre of the chamber (between inlet and outlet). Air speed at 1.7m
from the floor is shown at Figure 6.2a.
tCC
dpvdpi
dpi )()ln()0(,
, βλ +−=
221
To study the pattern of airflow, smoke generated by the Dyna-Fog Cyclone machine
and smoke candles were released inside the chamber, on separate occasions. While a
smoke candle is recommended in some studies (Persily, 2005), the fog machine has
produced a dense smoke which was easier to be sited and tracked than that of a candle
one. A typical sample of the airflow pattern for V1 is shown in Figure 6.2b, in which
the smoke rose up and gently moved toward the outlet. The smoke density in the
centre of the chamber (between the inlet and the outlet area) was greater than in the
areas close to the walls. V2 showed a similar airflow pattern, only with faster and
more turbulent smoke movements. The smoke at V0 also rose but at a very low speed
and it dispersed so as to give very similar densities throughout the chamber.
222
(a)
1 2 3 4
0.48 m0.96 m
1.44 m1.92 m
0
0.05
0.1
0.15
0.2
0.25
Door w all, 0.71 m distance from each measurement
Airs
peed
, m
/s
(b)
Figure 6.2: Air speed and smoke flow pattern at V1: (a) three dimensional view of air speed flow at level 1.7m; (b) Top view of the smoke pattern flow.
The temperature, relative humidity and the pressure inside the chamber were also
measured during the course of the experiments. The temperature and relative humidity
for the V0 experiments were 20.6-27.7oC and 43-60.7%, respectively. For V1 and V2,
they were 22-27oC and 43-55%. The average temperature outside the chamber was
223
less than that inside the chamber with a 1 ± 1oC variance. Vertically, the temperature
inside the chamber at 1.7 m was higher than that at 0.1 m (which is to be expected,
since hot air rises), however there was ≤ 0.3 oC variation across all measurement sites
within the chamber. Further, the chamber was under positive pressure when the
ventilation system was operating and the difference in pressure between inside and
outside the chamber was 0, 8 and 19 Pascals for V0, V1 and V2, respectively.
6.3.2 Deposition rate with the ventilation system off The concentration levels of the generated aerosols in this study were 3.4-5.5, 4.3-7.4,
10.2-17.4 and 8.4-19 particles/cm3 for Aspergillus, Penicillium, canola oil and talcum
powder, respectively, while the background for all measurements was 0.26-0.57
particles/cm3. Using the optical microscope, and based upon particle characteristics
such as size, shape and appearance, most of the bioaerosol particles were identified as
spores. Particles smaller than 1 µm were found also on the slides and were expected to
be fungal fragments. The resulting mode values for the background, Aspergillus,
Penicillium, canola oil and talcum powder were 0.97, 3.28, 2.29, 0.54 and 3.41 µm,
respectively, while their mean values were 1.18, 2.79, 2.14, 1.34 and 3.05 µm,
respectively. Figure 6.3 shows typical plots of particle concentration versus time, for
all particle types. After reaching peak concentrations, the decay rate stabilized for all
particle sizes in the four aerosols and plateaued prior to the end of the experiments
(not shown).
For comparison purposes, the sizes of the generated fungal particles were grouped
into four categories, (0.5-1µm, 1-2.5 µm, 2.5-5 µm and 5-6.3 µm), as shown in Table
6.2. These size ranges cover approximately 70-90% of viable fungi in indoor air (Li
and Kuo, 1994; DeKoster and Thorne, 1995). As shown in Table 6.2, the particle
deposition rate (h-1) increased with the increasing of particle aerodynamic diameters.
224
Figure 6.4 shows the particle deposition rate coefficients as a function of aerodynamic
diameter for all particle types, at the three different exchange rates. Each point in
Figure 6.4 represents the mean deposition rate of certain aerodynamic diameters in the
seven experiments. For the purpose of this study and for the clarity of Figure 6.4, the
standard deviations for particle deposition rate coefficients are presented separately in
Table 6.1. Figure 6.4 shows that the deposition rates increased with increasing particle
size for the aerosols and the bioaerosols under investigation.
225
(a)
1.72- 2.29 µm
0.001
0.010
0.100
1.000
10.000
0 2 4 6
Time, hour
Par
ticle
con
cent
ratio
n, #
/cm
3
Penicillium
C. oil
T. powder
(b)
2.46-3.52 um
0.001
0.010
0.100
1.000
10.000
0 2 4 6
Time, hour
Par
ticle
con
cent
ratio
n, #
/cm
3
Aspergillus
Penicillium
C. oil
T. powder
(c)
3.79-4.70 µm
0.001
0.010
0.100
1.000
10.000
0 2 4 6
Time, hour
Par
ticle
con
cent
ratio
n, #
/cm
3
Aspergillus
C. oil
T. powder
Figure 6.3: Typical particle concentration as a function of elapsed time for aerosols and bioaerosols under investigation, at V0 air exchange rate conditions.
226
(a)
0.01
0.1
1
10
0.1 1 10particle aerodynamic diameter, µm
Dep
ositi
on ra
te c
offic
ient
, h-1
Aspergillus
Penicillium
C. oilT. powder
(b)
1
10
0.1 1 10
Particle aerodynamic diameter, µm
Dep
ositi
on ra
te c
offic
ient
, h -1
Aspergillus
Penicillium
C. oil
T. powder
(c)
1
10
0.1 1 10
Particle aerodynamic diameter, µm
Dep
ositi
on ra
te c
offic
ient
, h -1
Aspergillus
Penicillium
C. oil
T. powder
Figure 6.4: Particle deposition rate coefficient, as a function of aerodynamic diameter, for aerosols and bioaerosols, at different air exchange rates: (a) V0 (airspeed = 0.01 m/s); (b) V1 (airspeed = 0.15 m/s); (c) V2 (airspeed = 0.30 m/s).
6.3.3 Deposition rates with the ventilation system on
Figures 4b and 4c show the particle deposition rate coefficients as a function of
aerodynamic diameter for the Aspergillus, Penicillium, canola oil and talcum powder
227
at V1 and V2, respectively. As with V0, the deposition rates at V1 and V2 increased with
increasing particle size. Figure 6.4 shows that particle deposition rates when the
ventilation system was turned on (V1) were greater than when it was turned off (V0),
and that a further increase in the ventilation rate (V2) caused even greater particle
deposition rates (Figure 6.4c). Table 6.2 shows that the increase in the ventilation
rates was followed by an increase in particle deposition rate.
02
468
101214
1618
0 0.5 1 1.5
Time, hour
Par
ticle
con
cent
ratio
n, #
/cm
3 Aspergillus
Penicillium
C. Oil
T. Powder
Figure 6.5: Typical particle concentration profiles as a function of time for the aerosols under investigation at V1 air exchange rate.
Figure 6.5 shows a typical particle concentration profile, as a function of elapsed time,
for the aerosols under investigation at V1. As for V0, the decay rate stabilized and
plateaued, for all particle sizes of the four aerosols, prior to the end of the
experiments.
228
Table 6-1: Standard deviations for particle deposition rate coefficients (h-1) (which are presented in Figure 6.4) as measured at different air exchange rates.
(V0) = 0.01 m/s V1= 0.15 m/s V2= 0.3 m/s
aDi. A P C T A P C T A P C T
0.542 0.01 0.69 0.95 0.777 0.01 1.02 0.83 1.33 1.037 0.01 1.12 0.94 1.09 1.286 0.02 0.80 1.04 1.03 1.486 0.02 0.03 0.68 1.20 0.90 0.95 0.85 1.596 0.03 0.02 0.95 0.53 0.85 0.96 0.67 1.715 0.04 0.04 0.04 0.65 0.49 0.64 1.20 0.45 0.59 1.843 0.03 0.05 0.09 0.78 0.87 0.59 1.42 1.10 0.78 1.981 0.02 0.02 0.02 0.91 0.69 0.48 0.95 0.69 0.68 2.129 0.04 0.07 0.02 0.48 0.89 0.69 0.78 0.85 0.49 2.288 0.02 0.06 0.04 1.17 0.59 0.79 0.68 0.75 0.93 2.458 0.04 0.05 0.04 0.06 0.82 0.49 0.48 1.00 1.30 0.76 0.95 1.32 2.642 0.11 0.02 0.13 0.10 0.67 0.38 0.72 0.59 0.98 0.59 0.68 0.74 2.839 0.11 0.03 0.05 0.09 0.48 0.79 0.45 0.75 0.87 0.48 1.11 0.72 3.051 0.07 0.05 0.06 0.08 1.09 0.88 0.39 0.33 0.65 1.20 0.68 0.59 3.278 0.08 0.10 0.11 0.09 0.47 0.65 0.66 0.56 0.98 0.68 0.84 0.83 3.523 0.05 0.07 0.06 0.09 0.75 0.69 0.85 0.76 1.36 0.82 0.76 3.786 0.11 0.08 0.06 0.69 1.30 0.48 0.82 0.74 1.07 4.068 0.06 0.05 0.19 1.10 1.06 0.69 0.59 0.86 0.67 4.371 0.12 0.08 0.24 0.73 0.68 0.57 1.42 0.91 0.54 4.698 0.13 0.09 0.05 0.52 0.61 0.48 0.92 0.64 0.65 5.048 0.04 0.17 0.76 0.92 1.19 0.78 5.425 0.12 0.33 0.53 0.86 0.68 0.82 5.829 0.13 0.27 0.45 0.75 0.79 0.91 6.264 0.14 0.24 0.42 0.72 0.89 1.28
aDi is aerodynamic diameter in µm and A, P, C and T stand for Aspergillus, Penicillium, canola oil and talcum powder, respectively.
229
Table 6-2: Particle deposition rate coefficients (h-1), for each of the four categories
0.5 - 1 (µm)
1 - 2.5 (µm)
2.5 - 5 (µm)
5 - 6.3 (µm)
Aerosol Ventilation Mean S.D Mean S.D Mean S.D Mean S.D Aspergillus V0 1.08 0.32 V1 4.59 0.61 V2 5.49 0.87 Penicillium
V0
0.08 0.02
0.3 0.10
0.71 0.17
V1 2.96 0.21 4.06 0.23 4.43 0.24 V2 5.44 0.52 6.36 0.78 C. oil
V0
0.07 0.03
0.22 0.09
0.94 0.27
2.66 0.41
V1 2.44 0.25 3.05 0.33 4.15 0.64 5.60 0.42 V2 3.86 0.21 4.68 0.57 5.59 0.56 7.79 0.59 T. powder
V0
0.43 0.14
1.48 0.48
3.35 0.26
V1 3.59 0.48 4.75 0.52 6.61 0.72 V2 5.35 0.41 6.30 0.72 8.52 0.62
230
6.3.4 Aerosol fluorescent percent Fluorescent particle number is the number of particles counted by the UVAPS in channels
2–64, while total particle number is the number of particles counted in the channels 1–64,
and the fluorescent percentage is the number of fluorescent particles as a percentage of
total particle number. The study of fluorescent percentage has added new information
about the auto fluorescing molecules present in the aerosol under investigation.
Figures 6a and 6b show the fluorescent percentage of investigated aerosols as a function
of elapsed time with the ventilation system off (V0) and on (V1), respectively. Both
figures show that all aerosols under investigation generated fluorescent signals. The
obtained fluorescent percentage results were 55 ± 3.1, 72.8 ± 1.69, 18.2 ± 1.69 and 11.9 ±
2.1% for Aspergillus, Penicillium, canola oil and talcum powder, respectively. As shown
in Figure 6.6a, the time needed to return to the background concentration decreased with
a decrease of the initial fluorescent percentage of each aerosol. The fluorescent particles
for Aspergillus, Penicillium, canola oil and talcum powder were in the size range of 1.98-
5.83 µm, 1.84 -3.78 µm, 1.84-6.26 µm and 2.46-7.23 µm and the modes of these
fluorescent particles were 3.05 µm, 2.46 µm, 2.13 µm and 4.07 µm, respectively. Fungal
fragments were also detected for Aspergillus and Penicillium samples. These fragments
had a strong fluorescent percentage (compared to the low fluorescent percentage of
background samples, and the SDA agar, which gave no fluorescent signals) and were
located in the high intensity channels (compared to the background samples which
occupied the lower channels (2–3) (see Section 4.5.))(Kanaani et al., 2008).
(a)
231
R2 = 0.9731R2 = 0.9843R2 = 0.8806R2 = 0.7219
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10
Elapsed time, hour
Fluo
resc
ent p
erce
ntag
e, %
AspergillusPenicilliumC. oilT. powder
(b)
R2 = 0.9534R2 = 0.9763R2 = 0.9572R2 = 0.8993
0
10
20
30
40
50
60
70
80
0 0.5 1
Elapsed time, hour
Fluo
resc
ent p
erce
ntag
e, %
AspergillusPenicilliumC. oilT. powder
Figure 6.6: Typical particle fluorescent percentage as a function of time: (a) air exchange rate at V0 (AER = 0.009 ± 0.003 h-1); (b) air exchange rate at V1 (AER= 1.75 ± 0.32 h-1).
6.4 Discussion
6.4.1 Air exchange rates, air speed and airflow patterns The obtained AER results were 0.009 ± 0.003, 1.75 ± 0.32 and 2.50 ± 0.18 h-1 for V0, V1
and V2, respectively. These values were within the ranges of many previous indoor
environmental studies. For example, the AER in most of the 64 elementary and middle
school classrooms in Michigan was found to be within this range (<3.0 h-1) (Daisey et al.,
232
2003). Wålinder et al. (1998) also reported that the average AER for 12 schools was 1.9
h-1.
The mean air speeds, as estimated in this study, were 0.01±0 .01, 0.15 ± 0.10 and 0.30
±0.12 m/s for V0, V1, and V2, respectively. These values were also within the ranges of
many previous indoor environmental studies. For example, Thorshauge (1982) found that
air speeds in indoor environments, such as offices and commercial places, were within the
range 0.05-0.40 m/s. Matthews et al. (1989) also found that the median air speeds in four
typical residential houses were 0.02-0.06 m/s when the central fan was off and 0.06-0.16
m/s when it was on.
In terms of airflow patterns, the smoke generated in the chamber was dispersed within a
few seconds after release, indicating a good level of mixing (Persily, 2005). Figure 6.2b
shows that the smoke moved in different directions due to the louvered inlet opening,
which diffused the air in all directions (360o). Visual inspection revealed the smoke
density in the middle of the chamber (between the inlet and the outlet area) was higher
than for the areas close to the walls, due to the suction of air through the outlet. As
expected, V2 showed faster and more turbulent smoke movements than at V1. The
distribution of airspeed in the chamber at V1 (Figure 6.2a) was consistent with the smoke
airflow patterns shown in Figure 6.2b, for the same ventilation rate.
The difference in pressure between the inside and outside the chamber was 0, 8 and 19
Pascals for V0, V1 and V2, respectively. These results fall within the ranges of typical
indoor/outdoor pressure differences (2–20 Pa) (Mosley et al., 2001). Bearg (2001) also
reported that the typical positive pressure in the occupied area of a building, with respect
to outdoors, is 12.5 Pa during the operation of mechanical ventilation system.
233
6.4.2 Fungal spore concentration levels While the concentration levels of the generated Aspergillus and Penicillium in this study
were between 3.4 x 106 - 5.5 x 106, and 4.3 x 106 - 7.4 x 106 particles/m3, respectively, the
indoor airborne fungi concentrations reported in existing literature were significantly
lower. Hargreaves et al. (2003b) reported that the concentrations of airborne fungi in
environmental samples measured indoors in 14 residential suburban houses in Brisbane
were 810 ± 389 CFU/m3 and a study by Lee et al. (2006) reported that the concentration
of airborne fungi in 6 Cincinnati homes was typically between 0 - 1362 CFU/m3. Under
heavy fungal colonization, in crawl space in Finland, airborne spore concentrations were
found as high as 103-104 CFU/m3 (Kurnitski and Pasanen, 2000). Hyvärinen (2000)
reported that the concentration range of viable fungi in indoor environments in Finland
was in the range 101-105 CFU/m3. As it can be seen from the above data, the maximum
airborne spore concentration was 1 x 105 CFU/cm3, which is less than the background
reported in this study (2.6 x 105 particles/m3). For this study, the concentrations of
Aspergillus and Penicillium were chosen in the following ranges, 3.4 x 106 - 5.5 x 106,
and 4.3 x 106 - 7.4 x 106 particles/m3 (with minimum viable particle of 1.8 x 106 – 3.0 x
106, and 3.1 x 106 – 5.4 x 106 particles/m3), respectively, so that the concentration of
fungal particles was more than ten times the background concentration for each
experiment. These concentrations are still considerably higher than those found in indoor
environments, however it was not reasonable to set them lower (at levels similar to those
found in indoor environments), otherwise the level of uncertainty would have been very
high.
6.4.3 Deposition rates and comparison with other studies when the ventilation system was off
The deposition rates in all experiments were calculated after complete mixing of the air in
the chamber was achieved. In each case, the deposition rates were calculated by
234
subtracting the losses due to AER for V0 (0.009 h-1), V1 (1.75 h-1) and V2 (2.5 h-1) and the
UVAPS sample flow rate (0.02 h-1) from the total particle loss rate. Aspergillus and
Penicillium showed similar decay when compared to canola oil and talcum powder,
which indicates the same behavior between the bioaerosols and non-biological aerosols
under investigation (Figure 6.3). The slight difference between the two types of aerosols
may be due to the different shapes and densities of the aerosols. As shown in Figure 6.4a,
the highest reported particle deposition rates were for talcum powder and the lowest were
for canola oil, while the deposition rates for Aspergillus and Penicillium fell somewhere
in between the two. Those low standard deviations that were shown in Table 6.1 reflect
the consistent results for the seven runs which were conducted for each of the aerosols.
Figure 6.4 shows the increase in particle deposition rate with an increase in the size of the
aerosols under investigation. The particle deposition rates for Aspergillus and Penicillium
showed similar values as for canola oil and talcum powder, which is indicative of the
similar behavior of these non-biological aerosols and fungal spores of the same size.
Aspergillus and Penicillium also showed a very similar deposition rates to each other,
which may be due to their similar densities and shapes (subglobose) (Raper et al., 1965;
Ramirez, 1982). The slight difference in deposition rates between the aerosols under
investigation may be due to differences in their densities and shapes. While fungal spores
where subgloboses with a density of 1.1 g/cm3 (Grinshpun et al., 2005), the canola oil
was spherical with a density of 0.91 g/cm3 and the talcum powder particles were irregular
with bulk density of 0.87 g/cm3 (both the shape and density of the canola oil and talcum
powder were determined in the laboratory using an electronic balance (Mettler PM 400)
and a light microscope (Model CX31RTSF, Olympus Corporation, Tokyo, Japan). The
aerosols under investigation showed statistically significant correlations between particle
aerodynamic diameter and deposition rate (r = 0.95, p < 0.0001 and r = 0.94, p < 0.0001, r
235
= 0.96, p< 0.0001 and r = 0.97, p <0.0001) for Aspergillus, Penicillium, canola oil and
talcum powder, respectively.
Particles of submicrometer sizes were detected for Aspergillus and Penicillium aerosols
and were deemed to be from fungal fragments (Section 4.4.). Fragmentation of fungal
spores was also reported by Górny et al. (2002) and Kildesø et al. (2003). While the
deposition rates of these submicron particles were calculated for Penicillium, they were
not calculated for Aspergillus because there were not enough fragments generated to
statistically analyse them, without a high level of uncertainty (Figure 6.4).
In order to compare the results of fungal deposition rates from this study with other
published findings, relevant studies on indoor environments were identified and
compared (Figure 6.7). These selected studies investigated non-bioaerosol particle
deposition rates in indoor environments, such as controlled test houses, chambers and in
residential houses. Most of these studies, likewise this study, were conducted under
normal ventilation conditions or in sealed chambers (with no mechanical ventilation). As
was shown in Figure 6.7, the concentrations of the investigated fungal spores and
fragments were comparable to those found in the other studies, with the deposition rates
shown as a function of particle size. The results for Aspergillus and Penicillium indicated
that the behaviour of these particles depended on their physical properties and that their
biological properties had no effect on their deposition rates (Figure 6.7a).
The results of this study were similar to other studies which were conducted under similar
conditions (for example, see Thatcher et al. (2002) in Figure 6.7a). The agreement
between the particle deposition rates in these two studies is likely to be due to the
similar: AER (0.009 ± 0.003 h1 for this study and 0.006 ± 0.003 h1 for Thatcher et al.
(2002)); volume to surface area ratio (2.23 for this study and 2.48 for the other); and use
236
of the unfurnished chamber. Figure 6.7a shows that the deposition rates of aerosols or
fungal spores were a function of particle size. For instance, for this study and that of
Thatcher et al. (2002), the deposition rate of 4.7 µm particles was more than 25 times
higher that for 0.54 µm particles.
6.4.4 Deposition rates and comparison with other studies when the ventilation system was on
Figure 6.4a, b show that the increase in air speed was followed by an increase in particle
deposition rate for Aspergillus, Penicillium, canola oil and talcum powder. The study
showed statically significant association between particle aerodynamic and deposition
rate at both V1 and V2 (r = 0.91, p < 0.005 and r = 0.90, p < 0.005, r = 0.92, p < 0.005 and r = 0.95, p
< 0.005 for Aspergillus, Penicillium, canola oil and talcum powder, respectively at V1 and r
= 0.89, p < 0.005 and r = 0.87, p < 0.005, r = 0.93, p < 0.005 and r = 0.94, p < 0.005 for Aspergillus,
Penicillium, canola oil and talcum powder at V2, respectively). Further statistical analysis
(Mann-Whitney U test) demonstrated that there were significant differences (p < 0.0001)
between the particle deposition rate of all aerosols under investigations when measured at
two different ventilations (V0 and V1).
This result agrees with previous published studies using small instrument-cooling fans or
ceiling fans (Mosley et al., 2001; Thatcher et al., 2002; Schnell et al., 2006), and also with
those using central heating and air conditioning fans (Xu et al., 1994; Howard-Reed et al.,
2003; Wallace et al., 2004a). However, Bouilly et al. (2005) found that an increase in
ventilation rate (using a mechanical ventilation system fan) did not lead to higher particle
deposition rates. While the previous studies were conducted on non-biological aerosols,
this work investigated the deposition rates of fungal particles and for the first time, in
indoor environments similar to that in offices and bedrooms.
237
Using small fans, Thatcher et al. (2002) found that the increase in the air speed from <2
cm/s to 19.1 cm/s caused an increase in the deposition rates by an average factor of 1.5
and 2.0 for particles with diameters <1.0 and larger particles, respectively. Schnell et al.
(2006) reported that the particle deposition velocity under stirred conditions (using a
centrifugal blower fan) in a 1.6 m3 chamber were 3.8–6.6 times higher than those under
still conditions.
To compare our results for fungal spores with the results of other studies using MVS, the
particle deposition rate coefficients (h-1) were grouped in four categories (0.5-1µm 1-2.5
µm 2.5-5 µm and 5-6.3 µm), as shown in Table 6.2. As in previous studies (Xu et al.,
1994; Howard-Reed et al., 2003; Wallace et al., 2004a), the particle deposition rates
increased with each size category. It was also found that particle deposition rates
increased further when the ventilation rate was increased from V1 to V2 (Figures 4b, c).
However, the increase in particle deposition rates found in this study, as a result of the
shift from V0 to V1, was greater than that found in the previous studies that measured
particle deposition rates with the ventilation system off and on. In this study, it was also
found that the increase in the deposition rates for smaller particles was greater than the
increase for larger particles.
For instance, the increase in particle deposition rates obtained for particles in the size
range 2.5-5 µm for Aspergillus, Penicillium, canola oil and talcum powder were 4.3, 6.2,
4.4 and 3.2 times, respectively, while Howard-Reed et al. (2003) has found a less
significant increase (2.5 times) for the same size range of particles when the HVAC fan
was turned on. This may be due to the difference between the AER’s for each study, in
that the AER increased 194 times in our study and just 7.8 times in Howard-Reed et al.
(2003) when the MVS was switched on. While the AER in this study was 0.009 h-1 at V0
238
and 1.75 h-1 at V1, it was 0.64 h-1 and 5 h-1 when the ventilation system was turned off
and on, respectively, in Howard-Reed et al. (2003).
The finding that the increase in particle deposition rates from V0 to V1 was greater for
smaller particles than for larger particles, can be explained by the fact that particle
movements in ventilated areas are strongly influenced by air flow patterns (Zhao et al.,
2004b) and also by the locations of the inlet diffuser and outlet (Bouilly et al., 2005).
When the AER was increased, the movement of smaller particles towards the walls
increased, so that collisions with the walls also increased and thus their deposition rates
increased too.
The particle deposition rates of larger fungal particles in this study were closer to those
found in previous studies (Xu et al., 1994; Emmerich and Nabinger, 2001; Howard-Reed
et al., 2003; Wallace et al., 2004a) than the deposition rates for smaller particles (Figure
6.7b).
It can be seen from Table 6.1a and Figure 6.4b that the increase in the deposition rates at
high AER’s (V1 and V2) was not uniform according to particle size (i.e., the deposition
rate of Penicillium at V1 was 4.30 h-1 for particles 1.84 µm in diameter and 4.09 h-1 for
particles 1.98 µm in diameter). Zhao et al. (2004b) also obtained similar results for
comparable sizes, indicating that the inertia created by the larger velocity air flows may
exceed the difference in gravitational pull exerted on the different particle sizes.
However, as expected, an overall increase in airflow rate in the chamber resulted in
decreased particle concentration (Krüger and Kraenzmer, 1996).
239
(a)
0.01
0.1
1
10
0.1 1 10Particle aerodynamic diameter, µm
Dep
ositi
on ra
te c
offic
ient
, h
-1
Aspergillus (Thisstudy)Penicillium (Thisstudy)Thatcher et al.,2002Fogh et al., 1997
Byrne et al., 1995
Vette et al., 2001
Thatcher &Laylon, 1995Mosley et al.,2001long et al., 2000w interHe et al., 2005
How ard-Reed etal., 2003Ferro et al., 2004
Wallance et al., 2004Long et al., 2000summer
(b)
0.1
1
10
0.1 1 10Particle aerodynamic diameter, µm
Dep
ositi
on ra
te c
offic
ient
, h -
1
Aspergillus (Thisstudy, AER = 1.75 /h)
Penicillium (Thisstudy, AER = 1.75 /h)
Wallace et al., 2004(AER = 5.4 /h)
How ard-Reed et al.,2003 (AER = 5.0 /h)
Xu et al., 1994 (3070RPM, AER = 0.02 /h)
Emmerich and Nabinger,2001 (AER = 0.22 /h)
Figure 6.7: A comparisons of particle deposition rates measured in houses and experimental chambers reported in literature and those found in this study: (a) no ventilation, where V0 represents this study; (b) ventilation or fan on, where V1 represents this study.
240
6.4.5 Aerosol fluorescent percent All aerosols under investigation generated fluorescent signals when the ventilation system
was both off and on (Figure 6.6). The fluorescent percentage results were 55 ± 3.1, 72.8 ±
1.69, 18.2 ± 1.69 and 11.9 ± 2.1% for Aspergillus, Penicillium, canola oil and talcum
powder, respectively. In spite of the fact that the UVAPS was designed as an aerosol
counter for the detection of viable airborne microorganisms (TSI-Incorporated, 2000), the
canola oil and talcum powder also gave strong fluorescent signals (Figure 6.6a, b). It has
also been reported in other studies that the agar washing, peptone water and broth media
also produced relatively strong fluorescent signals (Agranovski and Ristovski, 2005).
These results suggest that the UVAPS is not as selective for the specific microbial
fluorescent molecules as was intended (Hariston et al., 1997; TSI-Incorporated, 2000).
As shown in Figure 6.6, the fluorescent percentage decay at V0 and V1, as a function of
time, had very high R2 correlations for the slopes of Aspergillus and Penicillium (> 0.95)
and high R2 correlations (around 0.90) for canola oil, while it was less for talcum powder
(reaching 0.72 at V0). From these results it can be concluded that the fluorescent
molecules were distributed more homogenously for Aspergillus and Penicillium than that
for the other samples. The time that was needed for the fluorescent percentage to decline
to the background level (1.2 ± 0.7%) decreased as the initial fluorescent percentage of
each aerosol decreased. At V0, it was 9.5, 6.1, 3.9, 2.0 hours for Penicillium, Aspergillus,
canola oil and talcum powder, respectively (Figure 6.6a). The size of particles which
contain the fluorescent compound also affected the decay rate. For example, large
Aspergillus and Penicillium particles, which were deposited first, had a greater
fluorescent percentage than the smaller particles (Kanaani et al., 2007). A combination of
aerosol distribution, together with the initial fluorescent percentages, caused the
241
fluorescent percentage loss rate to be in the following descending order: Aspergillus,
Penicillium, talcum powder and canola oil.
While it took hours for the generated particles to reach the fluorescent background level
for V0, it took less than an hour for V1 (Figure 6.6b) (and even less for V2, figure not
shown) due to the effect of the ventilation on the particle deposition rates.
The real-time UVAPS instrument is a good tool for the investigation of the fungal
particles. Under controlled conditions, it identifies and confirms the species of fungi
under investigation through their size, fluorescent percentage and fluorescent intensity
(Kanaani et al., 2007). For instance, the submicron particles which were detected for
Aspergillus and Penicillium aerosols, were proven to be fungal fragment propagules as
follows: 1) they still displayed a strong fluorescent percentage; 2) the fragments appeared
in the high intensity channels; and 3) the overall fluorescent percentage of the
Aspergillus and Penicillium samples with the fragments at the peak concentration was
less (55 ± 3.1%, 72.8 ± 1.69%, respectively) than the samples without the fragments (61
± 2.7% and 81.2 ± 1.43%, respectively). The results focusing on spore fragmentation as a
function of air speed will be presented in more detail in a separate manuscript.
6.5 Conclusion
Deposition rates were determined at different air exchange rates (0.009, 1.75 and 2.50 h-
1), by generating Aspergillus, Penicillium, canola oil and talcum powder particles, in a
chamber simulating a typical indoor environment. The tested aerosols covered a wide
range (0.54-6.26 µm) of inhaled particle sizes and the study has proven that the
deposition rate of fungal particles (mostly spores) is a function of their aerodynamic
diameter, since the deposition rates of similar sized spores and non-biological aerosols
were very close to each other. The deposition rates of the bioaerosols (Aspergillus and
242
Penicillium) were found to be in the same range as found for non-biological particles in
other studies, especially for those conducted under similar conditions. Particle deposition
rates increased at higher ventilation rates and were a function of both the particle size and
AER. It was also found that the real-time UVAPS instrument is a good tool for the
investigation of fungal particles, however it was not found to be selective for bioaerosols
only (the purpose for which it was designed). The results also show that ventilation is an
important factor in reducing the residence time of these particles indoors. In conclusion,
knowing the deposition rates of fungal particles will assist greatly when studying the
dynamics of airborne fungi, as the remaining aerosol is essentially a function of their size.
Acknowledgements
This project was supported by Grant 1-9311-8174 from the Research Triangle Institute,
PO Box 12194 Research Triangle Park, NC 27709-2194. Special thanks go to Dr David
Ensor, Aerosol Science and Nanotechnology, whose vision made this study possible.
243
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CHAPTER 7 : General Discussion
7.1 Introduction
Concern regarding the health effects of indoor air quality has grown in recent years, due
to the increased prevalence of many diseases, as well as the fact that many people now
spend most of their time indoors. Consequently, bioaerosol research has also become a
topic of interest amongst scholars and practitioners concerned about indoor air quality
control, as well as the associated health effects and medical treatments. In addition,
bioaerosol research has also become a topic of scientific interest due to the increased
threat from biological weapons and bioterrorism. In contrast to the numerous studies
which have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in
indoor environments are still poorly understood. For example, few studies have focused
on fungal spore release or the deposition of fungal propagules in indoor environments.
The real-time detection and identification of bioaerosols has become desirable in
scientific laboratories, as well as medical and agricultural areas. During the last decade,
experiments have been conducted using the numerous instruments capable of measuring
fluorescence spectra, in order to determine which instruments were suitable for the real-
time detection of single viable airborne bioaerosols. As a result of these experiments, the
Ultraviolet Aerodynamic Particle Sizer (UVAPS) became the first commercially
produced instrument designed to detect airborne bioaerosols in real-time. While many
studies have used the UVAPS to detect airborne bacteria, only very limited research has
been done to evaluate the performance of UVAPS when monitoring airborne fungi. For
example, the factors which affect the performance and ability of the UVAPS to monitor
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fungi and its ability to distinguish between two fungal species are yet to be studied.
Therefore, investigations into the above, as well as the study of the dynamic of fungi and
fungal fragmentation as a function of many variables, were the primary objectives of this
project.
7.2 Principal significance of the findings
The results obtained from investigations into the dynamics of fungal propagules,
including fungal release and deposition, as well as the mechanisms of fungal spore
fragmentation, significantly improves scientific understanding of the fate of fungal
propagules in indoor air, as well as their deposition in the respiratory tract. The results
presented in this thesis also provide new knowledge about the performance of the
UVAPS when measuring viable airborne fungi, particularly in terms of the instrument
ability to distinguish between fungal species under controlled conditions. These findings
also enhance fundamental knowledge in relation to the instruments application, as well as
its capabilities and limitations. Hence, this work may also contribute to the development
of a new and improved UVAPS instrument, which is able to monitor and measure
bioaerosols with greater accuracy and efficiency. When combined, this new knowledge
on both UVAPS performance, as well as the behaviour of airborne fungal propagules,
will assist in improving technologies for the real-time monitoring of bioaerosols and
contribute in many other bioaerosol applications.
The particular aspects studied, as well as some of the major findings of this research are
summarised below (in the order in which the findings were published):
The UVAPS was chosen for the real-time monitoring of fungal aerosols for two reasons:
firstly, it was actually designed to detect bioaerosols, including fungi; and secondly, it
was tested and found to be effective for monitoring bacteria (with some restrictions).
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Initial investigations into the performance of the UVAPS for monitoring fungal aerosols,
under controlled laboratory conditions, first required a method for generating fungal
aerosols to be determined. The first method investigated used the Collison nebuliser,
which was found to be unable to generate fungal aerosols with diameters of 5 µm or
larger, and the concentration of the aerosols produced fluctuated significantly, as a result
of the non-homogeneous distribution of fungal aerosols in the solutions. In contrast, the
direct (dry generation) method, in which fungi were aerosolised directly from the cultures
growing on the agar plates, was found to be a simple, inexpensive, reproducible and easy
to control, and therefore, this method was chosen for most of the experiments conducted
as part of this work.
Subsequent experiments found the UVAPS to be sufficiently sensitive for detecting the
fluorescent biomolecules present in fungal spores. However, since this was the first time
the UVAPS was used for detecting and monitoring fungal spore concentrations, the
results had to be compared with the results of a standard sampler. These comparisons
showed that the results obtained from the UVAPS were comparable with the results
obtained from the AGI-30 impinger, which was used as a reference sampler. In addition,
the upper detection limit of the UVAPS for fungal aerosols was also investigated. A
linear relationship was observed for total particle and fluorescent particle concentrations
measured by the UVAPS and the instrument upper detection limit was shown to be
approximately 7 × 107 particles/m3. These findings, which demonstrated, for the first
time, that the UVAPS was capable of being used for monitoring fungal aerosols, allowed
for further experiments to be conducted, which used the UVAPS for investigations into
fungal spore behaviour.
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Experiments were also conducted in order to determine the ability of the UVAPS to
differentiate between genera (Aspergillus and Penicillium), under controlled laboratory
conditions. For example, the natural auto-fluorescence of fungal particles, which results
from the biochemical fluorophores found in the reduced fluorescent coenzymes NADH
and NADPH, as well as from the metabolic function riboflavin (which are present in the
viable cells of most organisms, including fungi), was investigated under a variety of
conditions. The results showed that the fluorescent fungal spore percentage decreased
with increasing fungal spore age, as well as with the number of times the fungal spores
were exposed to air currents. Fungal spore size of the genera under investigation was also
found to increase with increased culturing time. The size distribution of the spores was
also used as an additional parameter for differentiating between genera of the same age,
however it was not useful for differentiating between genera of different ages. Based on
fungal spore size distributions, together with fluorescent percentages and intensities, the
UVAPS was found to have the ability to discriminate between two fungal spore species,
under controlled laboratory conditions. These findings may help in establishing a method
for differentiating between fungal species under controlled conditions, as well as
providing important information for conducting intrinsic fluorescence studies, especially
those related to fungal spores. In the field, however, it would not be possible for the
UVAPS to differentiate between different fungal spores due to the presence of other
micro-organisms of varying age having been subjected to different environmental
conditions.
Fungal fragmentation mechanisms were also investigated using the UVAPS, in order to
determine how the instrument would respond to submicrometer fungal fragments, as well
as to gather further information about the behaviour of fungi in the air. Fungal fragments
were detected for all three of the species under investigation (Penicillium, Aspergillus and
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Cladosporium) when using the direct method of generation, with a wide range of air
velocities (1.8, 3.3, 5.3, 7.1, 8.5, 10.2 m/s). Fungal propagules were also detected at 1.8,
3.3 and 5.3 m/s using the fan and FSSST methods. It was also found that no
fragmentation occurred for any of the fungal species or for any method of generation at
air velocities ≤ 0.4 m/s. This finding indicates under typical indoor ventilation conditions,
where the air velocity is ≤ 0.4 m/s, there would be no fungal spore fragmentation for the
species under investigation, except that which occurs in the actual ventilation system,
where air speeds can reach up to 29.1 m/s. In addition, the study also showed a significant
correlation between fungal fragmentation percentage and air velocity for most of the air
velocities used in this study. These findings will not only help to improve the design of
mechanical ventilation systems, but they will also serve to improve air quality in indoor
environments.
The study also found that fungal spores were the specific part of the fungal colony that
underwent fragmentation. This finding will help in controlling fragmentation indoors, as
well as enhancing indoor air quality. Other results of the study also suggest that these
fungal spores either fragmented into two larger particles, along with many smaller
submicrometer particles or they fragmented into submicrometer particles only. Using the
UVAPS, submicrometer particles (which have a greater impact on health) were found to
increase by up to 400 times, 9.4 times and 6.3 times during fragmentation, for
Penicillium, Aspergillus and Cladosporium, respectively. These findings will assist in
understanding the nature of spores and their shells, and they also highlight the importance
of understanding fungal fragmentation methods, in order to reduce their impact on human
health. When subjected to fragmentation, the fluorescent percentage of fungal samples
was also found to decrease, for all species and all methods of generation. This finding
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provides an important insight into the behaviour of the biomolecules in fungi, and will
help in improving the detection capabilities of the UVAPS.
The full range of fungal particles produced in both fragmented and non-fragmented
samples (for Penicillium and Aspergillus) were also analysed for the first time, using a
combination of the UVAPS and an SMPS. The concentration of ultrafine particle
fragments for both species were found to be very low compared to the rest of the size
range (0.1-0.737 µm) measured by the SMPS. While the results obtained from the
UVAPS and the SMPS were not identical for the same samples, it was found that the
UVAPS was more sensitive than the SMPS when measuring samples that contained small
particle concentrations.
The deposition rates of Aspergillus and Penicillium at different air exchange rates was
also investigated. It was found that the deposition rate of fungal particles (mostly spores)
in a chamber simulating a typical indoor environment was a function of their
aerodynamic diameter. Particle deposition rates also increased at higher ventilation rates
and were a function of both particle size and air exchange rate. The deposition rates for
fungal particles were also found to be in the same range as for non-biological particles of
similar size measured in other studies, especially for those conducted under similar
conditions (i.e. generating canola oil and talcum powder particles, in a chamber
simulating a typical indoor environment). The results also show that ventilation is an
important factor in reducing the residence time of fungal particles indoors. Knowing the
deposition rates of fungal particles will assist greatly when studying the dynamics of
airborne fungi and developing a better understanding of fungal spore dynamics in indoor
environments.
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7.3 Conclusions
1 - UVAPS performance in relation to fungal spores:
• In contrast to the Collison nebuliser, the dry generation method for producing
fungal spores was found to be simple, reproducible, inexpensive and easy to
control.
• The UVAPS was found to be sensitive enough to detect the fluorescent
biomolecules present in fungal spores and hence, it was appropriate for use when
monitoring fungal spore concentrations.
• Based on fungal spore size distributions, together with the fluorescent percentages
and intensities of fungal species, the UVAPS had the ability to discriminate
between two fungal spore species under controlled laboratory conditions.
• The UVAPS was not found to be selective for bioaerosols only (as it was designed
for).
2 - Fungal fragmentation mechanisms:
• While fungal fragments were detected for all three species under investigation at
generation velocities > 1.8 m/s, no fragmentation was found at air velocities ≤ 0.4
m/s.
• Fungal spores were found to be the specific parts of the fungal colony which
underwent fragmentation.
• The fluorescent percentage of fungal samples was found to decrease when
subjected to fragmentation.
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3 - Fungal particle deposition rates:
• Fungal particle deposition rates were found to be a function of their aerodynamic
diameters.
• The fungal particle deposition rates were also found to increase at higher
ventilation rates.
The findings of this study are useful for improving scientific understating of the
characteristics of indoor viable bioaerosols, as well as their behaviour in indoor
environments and the factors that influence the fungal fragmentation processes. This work
also provides important information about instrumentation and techniques for the real-
time monitoring of viable bioaerosols in indoor environments, and it highlights the
important factors that will influence their ability to differentiate between different
microorganisms.
7.4 Future work
In addition to estimating the deposition rates for Aspergillus and Penicillium at different
air exchange rates, further investigations into the dynamics of other fungal species (with
different fungal spore shapes) are still necessary, in order to develop a comprehensive
model for the transport of fungal spores in indoor air.
Although fungal spores were found to be the specific part of the fungal colony which
underwent fragmentation in a chamber simulating a typical indoor environment, the exact
mechanisms of the fragmentation process remain unclear. The effect of other factors, such
as vibration, on the fungal fragmentation process may also need to be investigated. In
addition, it also remains unclear as to whether other parts of the fungal colony have the
ability to undergo fragmentation under different conditions.
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This study also found that the fluorescence measured by the UVAPS was not selective for
airborne micro-organisms, since many other organic materials have the ability to emit
fluorescence. To improve the accuracy of the UVAPS, the signals produced by non-
biological particles need to be partitioned and estimated separately, which would require
a significant amount of further study. The accuracy and selectivity of the UVAPS could
also be improved by conducting further investigations into different techniques, such as
using multiple UV excitation wavelengths to create more than one fluorescence spectrum
for each species or adding certain compounds that bind specifically to micro-organisms,
so that the fluorescence spectra measured are specific to the micro-organisms only.
The lower detection limit of the UVAPS was found to be 0.01 particles/cm3, and
therefore, in order to study fungal aerosols at these low concentrations, it is necessary for
the UVAPS to be operated with a dedicated concentrator. As such, future work may focus
on finding a suitable method to measure low concentrations of micro-organisms in indoor
environments. In addition, the sensitivity of the UVAPS in the field also requires further
investigation.