A review on bioaerosol science, technology and engineering: current and beyond

1
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

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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
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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

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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
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Global Asthma Impacts
China
According to WHO estimates, 300 million people
suffer from asthma and 255, 000 people died of
asthma in 2005
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Pneumonia Death in Children lt 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
289179 Thompson et al., JAMA 2004 2921333
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
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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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Bio-Sampling

• The first critical step
• for monitoring, assessment, or control strategy
  for the biological inhalation exposure

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Air Sampling Techniques

• Impactors
• Liquid impingers
• Filters
• Electrostatic precipitator

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Impactors

• 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
It was used in anthrax investigation
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Principle of Collection by Impactor
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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

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Particle Collection of Portable Samplers
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Portable Microbial Impactors
Bio-Culture 120 L/min
Microflow 120 L/min
SMA MicroPortable 28.3/141.5 L/min
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Portable Microbial Impactors
RCS High Flow 100 L/min
Millipore Air Tester 140/180 L/min
SAS Super 180 180L/min
They have been used in military sites
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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
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Sampler Testing System
Yao, M. and Mainelis, G. Aerosol Sci. Technology,
2006, 401-13.
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, 401-13.
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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, 3138
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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
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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

BioSampler
Powerful aerosol-2-hydrosol sampling techniques
are needed
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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

• 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

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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, 37513-527
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Physical Collection Efficiency of Electrosampler
Electrostatic field may have the ability to
collect viruses
Electrostatic field, 5kV/cm, was used
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Comparisons of outdoor bacteria sampling using
Electrosampler and BioStage impactor
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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Spanish Flu in 1918
In 1918, Spanish flu killed 675,000 people in
the U.S. and an estimated 2050 million people
worldwide
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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.
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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

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ELISA Sample Processing
Dissolve into 1.5 mL PBS 0.05 Tween 20
shaking 2 h
Dust Sieving (gt30 mg)
centrifuge 20 min
supernatant
96-well plate
Antibody coated 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
LAL
Activates
Endotoxin (LPS)
(1,3)-ß-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)
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LAL/Glucatell ???????
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)ALog(X)
60-80 min
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Results for Road Dust
In collaboration with Lovelace Respiratory
Research Laboratory
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Results for Road Dust
In collaboration with Lovelace Respiratory
Research Laboratory
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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
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qPCR for quantification of microbial species
qPCR reaction mixture
Template DNA
Forward/Reverse Primers, Probes
dNTPs
DNA Polymerase
Buffer
Tris, KCl, Mg2 , BAS , etc.
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qPCR Application Curves
Vesper et al, 2005, American Laboratory, pp. 11-12
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Quantification of Environmental Sample
Eff10(-1/slope) -1
XnX0(1E)n
qPCR can be used for detecting airborne low
concentration biological agents
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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, 393338-3344
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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, 393338-3344
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Use of nano-scale Zero Valent iron particles in
Inactivating Microbial Species
Characterization of nanoscale iron particles

• FE-SEM Images
• NZVI particles
  Iron Oxides

Shaking-oxidization
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Inactivation of B. subtilis by NZVI particles
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Characterization of the Inactivation of B.
subtilis

(a)
(b)
Pure BST
NZVIBST anaerobic
(c)
(d)
FeOOHBST Aerobic
NZVIBST Aerobic

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Inactivation of P. fluorescence by NZVI
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Inactivation Mechanisms by NZVI
Gram-positive
Matthew T. Cabeen Christine
Jacobs-Wagner,Nature Reviews Microbiology 3,
601-610 (August 2005)
Gram-negative
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Inactivation of A. versicolor by NZVI
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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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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

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Biological Exposure Assessment and Control
Air Sampling
Bioaerosol Emission
Exposure Assessment
Detection
Biological Control
Prevent
Minimize
Human Biological Exposure
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Biological Exposure Assessment and Control
Detect
Control
Collect
CDC
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Bioaerosol field is multidisciplinary , and
requires many areas of expertise
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Thank you !!!
Maosheng Yao,PhD PKU 100 Scholar Program
Professor Email yao_at_pku.edu.cn Web
http//pantheon.yale.edu/my227/