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

Maosheng Yao

College of Environmental Sciences and EngineeringPeking University

Outline

Bioaerosol Emissions

Air Sampling Techniques

Biological Assessment, Detection and Control

Current State of Bioaerosol Research

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

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

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

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

Pneumonia Death in Children < 5 Years Old

WHO estimates that up to 1 million children under 5 die each year from pneumonia.

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

Global Outbreak of SARS in 2003

Social Impacts of SARS

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

The first critical step

for monitoring, assessment, or control strategy for the biological inhalation exposure

Bio-Sampling

Air Sampling Techniques

Impactors

Liquid impingers

Filters

Electrostatic precipitator

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

Principle of Collection by Impactor

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

Particle Collection of Portable Samplers

Microflow120 L/min

Bio-Culture120 L/min

SMA MicroPortable28.3/141.5 L/min

Portable Microbial Impactors

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

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

Sampler Testing System

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

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

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

Biological Collection Efficiency

the ability of the sampler to not only collect, but also keep the

viability of the bio-particles

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

Penicillium melinii, dae= 3.14m

Air sampling Techniques

filtration gelatin

filter

Anthrax surrogate

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

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

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

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

Physical Collection Efficiency of Electrosampler

Electrostatic field, 5kV/cm, was used

Electrostatic field may have the ability to collect viruses

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

Use of Electrostatic Field in Collecting Airborne Toxins

Use of Electrostatic Field in Collecting Airborne Allergens

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.

Globally confirmed human cases of H5N1 avian influenza since 2003

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

“In 1918, Spanish flu killed 675,000 people in the U.S. and an

estimated 20–50 million people worldwide”

Spanish Flu in 1918

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.

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

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

Procedure of ELISA tests

ELISA can be used together with air sampling technique for measuring airborne viruses and allergens

House Dust Mite (Der p & Der f 1)

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)

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

Results for Road Dust

In collaboration with Lovelace Respiratory Research Laboratory

In collaboration with Lovelace Respiratory Research Laboratory

Results for Road Dust

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:

qPCR for quantification of microbial species

qPCR reaction mixture:

Template DNA

Forward/ReversePrimers, Probes

dNTPsDNA

PolymeraseBuffer

Tris, KCl, Mg2+ ,

BAS , etc.

qPCR Application Curves

Vesper et al, 2005, American Laboratory, pp. 11-12

Sample19

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

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

Microorganism Inactivation

Yao and Mainelis, Environmental Science Technology, 2005, 39:3338-3344

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

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

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

Characterization of the Inactivation of B. subtilis

(a) (b)

(c) (d)

Pure BST

NZVI+BST Aerobic

NZVI+BST anaerobic

FeOOH+BST Aerobic

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

Matthew T. Cabeen & Christine Jacobs-Wagner， Nature Reviews Microbiology 3, 601-610 (August 2005)

Inactivation Mechanisms by NZVI

Gram-positive

Gram-negative

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

Characterization of the Inactivation of A. versicolor

Submitted to Applied & Environmental Microbiology

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

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

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

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

Biological Exposure Assessment and Control

BioaerosolEmission

Air Sampling

ExposureAssessment

Detection

Biological Control

Human Biological Exposure

Prevent Minimize

Collect Detect Control

CDC

Biological Exposure Assessment and Control

Bioaerosol field is multidisciplinary , and requires many areas of expertise

Thank you !!!

Maosheng Yao， PhDPKU “100 Scholar Program” Professor

Email: yao@pku.edu.cnWeb: http://pantheon.yale.edu/~my227/