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A review on bioaerosol science, technology and engineering: current and beyond
Maosheng Yao
College of Environmental Sciences and EngineeringPeking University
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Outline
Bioaerosol Emissions
Air Sampling Techniques
Biological Assessment, Detection and Control
Current State of Bioaerosol Research
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Particles of biological origins
--bacteria, fungi, viruses , poll
en --their derivatives such as en
dotoxins, glucans, and mycotoxins
--fungal allergens, indoor ho
use dust mites, dog, cat allergens
Biological Agents in the Environments
0.5-2μm for bacteria & 2-5μm for fungi commonly found, viruses are usually below 0.3 μm
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Bioaerosol Emissions
1) Natural environments2) Human and animal are sources of bioaer
osol 3) Waste recycling, bio-solid land applicatio
n, composting, agriculture, pharmaceutical and bio-tech activities
4) Hospital settings (surgery, organ transplant, and dental treatment)
5) Bioterrorism events
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Exposure to the biological agents presents a serious health challenge both for public and private sectors
Why Are We Concerned about These Biological Agents?
Respiratory diseases such as asthma, pneumonia, and allergies.
Infectious diseases such as SARS, and Bird Flu.
SARS outbreak in 2003
Influenza (bird flu) Outbreak
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Global Asthma Impacts
According to WHO estimates, 300 million people suffer from asthma and 255, 000 people died of asthma in 2005
USA
Brazil
Canada
Australia
China
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Pneumonia Death in Children < 5 Years Old
WHO estimates that up to 1 million children under 5 die each year from pneumonia.
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Annual Impacts of Epidemic Influenza
Estimates for the US
Cases: 20-50+ millions Days of illness: 100-200 millions Work & school loss: tens of millions Hospitalizations: 85,000-550,000 Deaths: 34,000-50,000 Economic loss: billions of dollars
MMWR 2003; 52 (RR-8); Thompson et al. JAMA 2003; 289:179Thompson 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
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Global Outbreak of SARS in 2003
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Social Impacts of SARS
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Increasing Threat of Bio-terrorism That May Release Lethal Airborne Biological Agents
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The first critical step
for monitoring, assessment, or control strategy for the biological inhalation exposure
Bio-Sampling
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Air Sampling Techniques
Impactors
Liquid impingers
Filters
Electrostatic precipitator
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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
--28.3 Liter/min with an impaction velocity of 24m/s
BioStage
Impactors
It was used in anthrax investigation
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Principle of Collection by Impactor
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Becoming more popular for sampling airborne biological agents
-- Battery-powered, portable, easy to handle
-- High volume sampling, more than 100 L/min
Portable Microbial Impactors
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Particle Collection of Portable Samplers
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Microflow120 L/min
Bio-Culture120 L/min
SMA MicroPortable28.3/141.5 L/min
Portable Microbial Impactors
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RCS High Flow 100 L/min
Millipore Air Tester 140/180 L/min
SAS Super 180 180L/min
Portable Microbial Impactors
They have been used in military sites
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MAS-100100 L/min
Portable Microbial Impactors
These samplers are increasingly being used for bio-sampling
Their sampling performances
are not fully described or investigated
Investigation of physical and biological collection efficiencies
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Sampler Testing System
Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.
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Physical Collection Efficiencies & Cutoff Sizes of Seven Portable Microbial Samplers When Sampling PSL Particles
Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.
anthrax
Virus
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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
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Biological Collection Efficiency
the ability of the sampler to not only collect, but also keep the
viability of the bio-particles
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Influences of Jet Velocity and Jet-to-plate Distance on Biological Collection Efficiency
Penicillium melinii, dae= 3.14m
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Air sampling Techniques
filtration gelatin
filter
Anthrax surrogate
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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
Powerful aerosol-2-hydrosol sampling techniques are needed
BioSampler
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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
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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
Electrostatic Collection
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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, 37:513-527
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Physical Collection Efficiency of Electrosampler
Electrostatic field, 5kV/cm, was used
Electrostatic field may have the ability to collect viruses
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Comparisons of outdoor bacteria sampling using Electrosampler and BioStage impactor
BioStage
ES
C
CR
Sampling Flow Rate, L/min
Co
nc
en
tra
tio
n o
f A
irb
orn
e C
ult
ura
ble
B
ac
teri
a, C
, NC
FU/ m
3
0
100
200
300
400
500
ElectrosamplerBioStage Impactor
Day # 1
0
200
400
600
800
1000
1200
1400
ElectrosamplerBioStage Impactor
Day # 2
R =5.7
R =2.1
R =1.8
R =4.8
R =2.2
R =2.5
1.2 28.3 10.0 28.328.35.0 1.2 28.3 28.3 28.310.05.0
Electrostatic method provides a better biological quantification
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Use of Electrostatic Field in Collecting Airborne Toxins
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Use of Electrostatic Field in Collecting Airborne Allergens
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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.
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Globally confirmed human cases of H5N1 avian influenza since 2003
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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
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“In 1918, Spanish flu killed 675,000 people in the U.S. and an
estimated 20–50 million people worldwide”
Spanish Flu in 1918
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Virusparticles
Air Out
E
Detection of influenza A Virus
Metal Plate
qPCR ELISA
Metal Plate Biosensor+
Air In
96-well-plate Endotoxin/Glucan
Yao et al. (2007) Integration of Technologies for Constant Monitoring of Exposure [E-Letter], Science.
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Environmental Allergens
Common Allergens? House dust allergens (Der p 1 and Der f 1), cat a
llergens 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
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ELISA Sample Processing
Dust Sieving (>30 mg)
Dissolve into 1.5 mL PBS
0.05% Tween 20
centrifuge20 min
shaking2 h
supernatant
96-well plate
Antibodycoated plate
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Procedure of ELISA tests
ELISA can be used together with air sampling technique for measuring airborne viruses and allergens
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House Dust Mite (Der p & Der f 1)
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Principle of LAL/Glucatell Assay(airborne endotoxin and glucan)
LAL
Factor C Factor G LAL
Factor B
Endotoxin (LPS)
LAL
LAL
Activates
Preclotting EnzymeLAL
(1,3)-β-D-glucan
LAL
pNA (yellow)
Substrate Ac-Ile-Glu-Ala-Arg-pNA.
Horseshoe crab LAL (Limulus Amoebocyte Lysate)
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LAL/Glucatell 分析方法的流程Filter
Extraction
0.05% Tween 20 for Endotoxin0.5 N NaOH for glucan, neutralized by Tris-HCL
Dilution (10-3)Add sample extracts,
standards into 96 well plate
Incubation (15 min at 37 oC)
add LAL or Glucatell Agents
Placed inside spectraphotometer
Log(Y)=A+Log(X)
60-80 min
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Results for Road Dust
In collaboration with Lovelace Respiratory Research Laboratory
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In collaboration with Lovelace Respiratory Research Laboratory
Results for Road Dust
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qPCR for quantification of microbial species
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
Primary tasks include:
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qPCR for quantification of microbial species
qPCR reaction mixture:
Template DNA
Forward/ReversePrimers, Probes
dNTPsDNA
PolymeraseBuffer
Tris, KCl, Mg2+ ,
BAS , etc.
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qPCR Application Curves
Vesper et al, 2005, American Laboratory, pp. 11-12
Sample19
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Quantification of Environmental Sample
qPCR can be used for detecting airborne low concentration biological agents
Eff=10(-1/slope) -1
Xn=X0(1+E)n
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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
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Microorganism Inactivation
Yao and Mainelis, Environmental Science Technology, 2005, 39:3338-3344
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Survival rates of P. fluorescens bacteria when deposited on MCE filter and exposed to the electrostatic field
Yao and Mainelis, Environmental Science Technology, 2005, 39:3338-3344
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Use of nano-scale Zero Valent iron particles in Inactivating Microbial Species
• FE-SEM Images
NZVI particles Iron OxidesShaking-oxidization
Characterization of nanoscale iron particles
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Inactivation of B. subtilis by NZVI particles
Unoxidized NZVI
FeO(OH) NZVI10mg/ml
NVZI1mg/ml
NZVI0.1mg/ml
Supernatant
Suvival Rate of BST
Nano Iron Suspension
Sur
viva
l Ra
te,
%
0
20
40
60
80
100
120
140
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Characterization of the Inactivation of B. subtilis
(a) (b)
(c) (d)
Pure BST
NZVI+BST Aerobic
NZVI+BST anaerobic
FeOOH+BST Aerobic
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Inactivation of P. fluorescence by NZVI
Survival Rate of PFL
Nano Iron Suspension
Sur
viva
l Rat
e, %
0
20
40
60
80
100
120
Unoxidized NZVI
FeO(OH) NZVI10mg/ml
NVZI1mg/ml
NZVI0.1mg/ml
Supernatant
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Matthew T. Cabeen & Christine Jacobs-Wagner, Nature Reviews Microbiology 3, 601-610 (August 2005)
Inactivation Mechanisms by NZVI
Gram-positive
Gram-negative
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Inactivation of A. versicolor by NZVI
Survival Rate of ASP
Nano Iron Suspension
Su
rviv
al R
ate,
%
0
20
40
60
80
100
120
140
Unoxidized NZVI
FeO(OH) NZVI10mg/ml
NVZI1mg/ml
NZVI0.1mg/ml
Supernatant
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Characterization of the Inactivation of A. versicolor
Submitted to Applied & Environmental Microbiology
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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
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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
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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
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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
Current Research Areas of BioAerosol Science
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Biological Exposure Assessment and Control
BioaerosolEmission
Air Sampling
ExposureAssessment
Detection
Biological Control
Human Biological Exposure
Prevent Minimize
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Collect Detect Control
CDC
Biological Exposure Assessment and Control
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Bioaerosol field is multidisciplinary , and requires many areas of expertise
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Thank you !!!
Maosheng Yao, PhDPKU “100 Scholar Program” Professor
Email: [email protected]: http://pantheon.yale.edu/~my227/