1. College of Environmental Sciences and Engineering
A review on bioaerosol science, technology and engineering: current and beyond
Maosheng Yao
College of Environmental Sciences and Engineering
Peking University

2. Outline Bioaerosol Emissions Air Sampling Techniques
Biological Assessment, Detection and Control
Current State of Bioaerosol Research

3. Biological Agents in the Environments
Particles of biological origins
--bacteria, fungi, viruses , pollen
--their derivatives such as endotoxins, glucans, and mycotoxins
--fungal allergens, indoor house dust mites, dog, cat allergens
0.5-2μm for bacteria & 2-5μm for fungi commonly found,
viruses are usually below 0.3 μm

4. Bioaerosol Emissions Natural environments
Human and animal are sources of bioaerosol
Waste recycling, bio-solid land application, composting, agriculture, pharmaceutical and bio-tech activities
Hospital settings (surgery, organ transplant, and dental treatment)
Bioterrorism events

5. Why Are We Concerned about These Biological Agents?
Exposure to the biological agents presents a serious health challenge both for public and private sectors
Respiratory diseases such as asthma, pneumonia, and allergies.
Infectious diseases such as SARS, and Bird Flu.
SARS outbreak in 2003
Influenza (bird flu) Outbreak

6. Global Asthma Impacts
USA
Brazil
Canada
Australia
China
According to WHO estimates, 300 million people suffer from asthma and 255, 000 people died of asthma in 2005

7. Pneumonia Death in Children < 5 Years Old
WHO estimates that up to 1 million children under 5 die
each year from pneumonia.

8. MMWR 2003; 52 (RR-8); Thompson et al. JAMA 2003; 289:179
Annual Impacts of Epidemic Influenza
Estimates for the US
Cases: millions
Days of illness: millions
Work & school loss: tens of millions
Hospitalizations: 85, ,000
Deaths: 34,000-50,000
Economic loss: billions of dollars
MMWR 2003; 52 (RR-8); Thompson et al. JAMA 2003; 289:179
Thompson et al., JAMA 2004; 292:1333; Adams PF et al. Vital Health Stat
1999; 10(200)
In addition, infectious diseases took a tremendous toll both on human and economy

9. Global Outbreak of SARS in 2003

10. Social Impacts of SARS

11. Increasing Threat of Bio-terrorism That May Release Lethal Airborne Biological Agents

12. The first critical step
Bio-Sampling
The first critical step
for monitoring, assessment, or control strategy for the biological inhalation exposure

13. Air Sampling Techniques
Impactors
Liquid impingers
Filters
Electrostatic precipitator

14. Impactors It was used in anthrax investigation
Andersen six-stage impactor was
developed in the 50s and
widely used as a standard for
bioaerosol sampling
BioStage Impactor (SKC, Inc., Eighty Four, PA)
--collect microorganisms onto agar surface
Liter/min with an impaction velocity of 24m/s
BioStage
Velocity is about 24m/s
It was used in anthrax investigation

15. Principle of Collection by Impactor

16. Portable Microbial Impactors
Becoming more popular for sampling airborne biological agents
-- Battery-powered, portable, easy to handle
-- High volume sampling, more than 100 L/min

17. Particle Collection of Portable Samplers

18. Portable Microbial Impactors
Bio-Culture
120 L/min
Microflow
120 L/min
SMA MicroPortable
28.3/141.5 L/min

19. Portable Microbial Impactors
RCS High Flow
100 L/min
SAS Super 180
180L/min
Millipore Air Tester
140/180 L/min
They have been used in military sites

20. Portable Microbial Impactors
These samplers are increasingly being used for bio-sampling
Their sampling performances
are not fully described or investigated
MAS-100
100 L/min
Investigation of physical and biological collection efficiencies

21. Sampler Testing System
Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.

22. Physical Collection Efficiencies & Cutoff Sizes of Seven Portable Microbial Samplers When Sampling PSL Particles
Virus
anthrax
Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.

23. Comparison of Sampler Performance with Particle Inhalation and Deposition in Human Lung
Yao and Mainelis, J. of Exposure Analysis and Env Epi, (2007), 17, 31–38

24. Biological Collection Efficiency
the ability of the sampler to not only collect, but also keep the viability of the bio-particles

25. Influences of Jet Velocity and Jet-to-plate Distance on Biological Collection Efficiency

26. Air sampling Techniques
filtration
gelatin filter
Anthrax surrogate

27. Air Sampling Techniques
BioSampler
1) Liquid Impinger, use of centrifuge and impaction to collect aerosol particles with a sampling flow rate of 12.5 L/min
2) Longer sampling time up to 8 hours
3) Transferring aerosols into hydrosols
BioSampler
Powerful aerosol-2-hydrosol
sampling techniques are needed

28. Bio- sampling Challenges
Impaction-based sampling techniques were shown to cause damages to the viability of microorganisms
Virus is too small to be collected by these techniques & their sampling method is significantly lacking
There is a need to develop a more advanced sampling strategy

29. Electrostatic Collection
Electrostatic collection is a mechanism of collecting the airborne charged particles using the electrical force
Collection velocity is about 2 to 4 orders of magnitude lower than that of BioStage impactor (24 m/s)
Lower mechanical stress and less desiccation upon the microorganisms being sampled

30. Yao and Mainelis, Journal of Aerosol Science, 2006, 37:513-527
Electrosampler was designed to investigate if natural charges of microorganisms can be used for their effective electrostatic collection
Yao and Mainelis, Journal of Aerosol Science, 2006, 37:

31. Physical Collection Efficiency of Electrosampler
Collection efficiency decreases with increase of flow rates
70% collection efficiency can be achieved at 1.2L/min
Larger particles have lower concentrations
Electrostatic field may have the ability to collect viruses
Electrostatic field, 5kV/cm, was used

32. Comparisons of outdoor bacteria sampling using Electrosampler and BioStage impactor
Electrostatic method provides a better biological quantification

33. Use of Electrostatic Field in Collecting Airborne Toxins

34. Use of Electrostatic Field in Collecting Airborne Allergens

35. Bioaerosol Detection and Assessment (Combining Physical , Biochemical and Molecular Techniques )
The electrostatic method demonstrates ability in collecting viruses from the air.
Virus concentration could be very low
in the air, even collected, might not be enough
to be detected.
Combination of electrostatic method with
advanced molecular techniques such as
qPCR and ELISA may offer a solution, e.g., for
detecting influenza A virus.

36. Globally confirmed human cases of H5N1 avian influenza since 2003

37. (magnified approximately 70,000 times) in May 1997
Influenza A Virus
Commonly known as flu, is an infectious disease of birds and mammals caused by an RNA virus
Typically, influenza is transmitted from infected mammals through the air by coughs or sneezes, creating bioaerosols containing the virus
Currently, the strand is only limited to animals, but it is very likely to mutate further becoming a human-to-human case.
Hong Kong Flu
(magnified approximately 70,000 times) in May 1997

38. Spanish Flu in 1918
“In 1918, Spanish flu killed 675,000 people in the U.S. and an estimated 20–50 million people worldwide”

39. Detection of influenza A Virus
Metal Plate
Virus
particles
Air Out
Air In E
qPCR
ELISA
Metal Plate
Biosensor +
96-well-plate
Endotoxin/Glucan
Yao et al. (2007) Integration of Technologies for
Constant Monitoring of Exposure [E-Letter], Science.

40. Environmental Allergens
Common Allergens?
House dust allergens (Der p 1 and Der f 1), cat allergens Fel d 1 (cat), dog allergen Can f 1 (dog), Bla g 1 (cockroach), Bla g 2 (mouse)
fungal allergens, e.g., Alternaria alternata allergen Alt1
Enzyme-Linked ImmunoSorbent Assay (ELISA) is often used to analyze allergens

41. ELISA Sample Processing
Dissolve into
1.5 mL PBS
0.05% Tween 20
shaking
2 h
Dust Sieving
(>30 mg)
centrifuge
20 min
supernatant
96-well plate
Antibody
coated plate

42. Procedure of ELISA tests
ELISA can be used together with air sampling technique
for measuring airborne viruses and allergens

43. House Dust Mite (Der p & Der f 1)

44. Principle of LAL/Glucatell Assay (airborne endotoxin and glucan)
Activates
Endotoxin (LPS)
(1,3)-β-D-glucan
Factor C
Factor G
LAL
LAL
Factor B
LAL
Preclotting Enzyme
LAL
Substrate Ac-Ile-Glu-Ala-Arg-pNA.
pNA (yellow)
Horseshoe crab LAL (Limulus Amoebocyte Lysate)

45. LAL/Glucatell 分析方法的流程
Add sample extracts,
standards into
96 well plate
Filter
Extraction
Dilution (10-3)
0.05% Tween 20 for Endotoxin
0.5 N NaOH for glucan, neutralized
by Tris-HCL
Incubation
(15 min at 37 oC)
add LAL or
Glucatell Agents
Placed inside
spectraphotometer
Log(Y)=A+Log(X)
60-80 min

46. Results for Road Dust
In collaboration with Lovelace Respiratory Research Laboratory

47. Results for Road Dust
In collaboration with Lovelace Respiratory Research Laboratory

48. qPCR for quantification of microbial species
Primary tasks include:
Selection or design of primer sets for specific microbial species (alternaria spp)
Design of probes for specific allergens & develop standard curves
quantitative-PCR tests for DNA extracts from environmental samples
DNA extraction
Primers & Probes
Standard Curve
qPCR tests

49. qPCR for quantification of microbial species
reaction mixture:
Template
DNA
Forward/Reverse
Primers, Probes
dNTPs
DNA
Polymerase
Buffer
Tris, KCl, Mg2+ , BAS , etc.

50. qPCR Application Curves
Sample 19
Vesper et al, 2005, American Laboratory, pp

51. Quantification of Environmental Sample
Eff=10(-1/slope) -1
Xn=X0(1+E)n
qPCR can be used for detecting airborne low concentration biological agents

52. Other Bioaerosol Detection Techniques
1) Bioaerosol mass spectrometry (BAMS) (Herbert et al., 2005)
2) Surface-enhanced Raman spectroscopy (Sengupta et al., 2007)
3) Flow cytometry with fluorochrome (Chen and Li, 2005
4) Bio-functional oligonucleotides based techniques such as aptamer (Brody et al., 1999)
5) Nanowire-based detection techniques
All these techniques can be also used for
detecting airborne low concentration biological agents

53. Microorganism Inactivation
> Bacteria Preparation In this research, we have tested both sensitive microorganisms and hardy microorganisms
(P. fluorescens and Bacillus subtilis )
> Deposition of Bacteria
on filters
>Exposure of filter-borne
bacteria
MCE filter (Mixed Cellulose Ester)
Yao and Mainelis, Environmental Science Technology, 2005, 39:

54. Survival rates of P. fluorescens bacteria when deposited on MCE filter and exposed to the electrostatic field
Survival rate of Pseudomonas fluorescens bacteria deposited on MCE filter and exposed to positive electrostatic field
Yao and Mainelis, Environmental Science Technology, 2005, 39:

55. Use of nano-scale Zero Valent iron particles in Inactivating Microbial Species
Characterization of nanoscale iron particles
FE-SEM Images
NZVI particles Iron Oxides
所有被表征的与微生物混合后的铁溶液都与细菌或者真菌悬浊液振荡混合5分钟。
Shaking-oxidization

56. Inactivation of B. subtilis by NZVI particles
关键！！[1] BST原来只有20%-30%灭活。 [2]

57. Characterization of the Inactivation of B. subtilis
Pure BST
NZVI+BST
anaerobic
(c)
(d)
FeOOH+BST
Aerobic
NZVI+BST
Aerobic

58. Inactivation of P. fluorescence by NZVI

59. Inactivation Mechanisms by NZVI
Gram-positive
Matthew T. Cabeen & Christine Jacobs-Wagner，Nature Reviews Microbiology 3, (August 2005)
Gram-negative

60. Inactivation of A. versicolor by NZVI

61. Characterization of the Inactivation of A. versicolor
Submitted to Applied & Environmental Microbiology

62. Summary of NZVI Inactivation
Inactivation was fast and efficient,
within 5 minutes, all B. subtilis were inactivated
Due to its small size, the inactivation efficiency was very high, and 10mg/ml can achieve good results
The inactivation depended on the membrane type of the microbial species, e.g., no effects on fungi species tested

63. Other Biological Control Technologies
Biofiltration (Sanchez-Monedero et al., 2003)
X-Ray enhanced electrostatic field
Photocatalytic materials such as TiO2 have been investigated (Pal et al., 2005).
Cold plasma (Birmingham and Hammerstrom, 2000) and UV light (Tseng and Li, 2005)
Control of air stream, e.g., negative pressure rooms

64. Current Research Areas of BioAerosol Science
Integration of bioaerosol science with molecular science such as qPCR, PCR, and ELISA
High volume of sampling: Portable Microbial Sampler, aerosol-2-hydrosol techniques
Investigation of the link between bioaerosol exposure and health effects
Development of high throughput environmental bio-sensor

65. Current Research Areas of BioAerosol Science
Combining bioaerosol with physics, chemistry, bio-medical engineering and molecular techniques
Drug delivery to the lung using aerosol technology
Human early disease detection such as lung cancer
Inactivation of BioAerosols

66. Biological Exposure Assessment and Control
Air Sampling
Bioaerosol
Emission
Exposure
Assessment
Detection
Biological
Control
Prevent
Minimize
Human Biological Exposure

67. Biological Exposure Assessment and Control
Detect
Control
Collect
CDC

68. Bioaerosol field is multidisciplinary ,
and requires many areas of expertise

69. Thank you !!! Maosheng Yao，PhD PKU “100 Scholar Program” Professor
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